KR101221186B1 - Polymeric beads for oligomer synthesis - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to solid support media for use in oligomer synthesis, methods of making the media, and methods of using the media. In some embodiments, the method of the present invention comprises the steps of (a) providing an organic phase comprising an olefin monomer, a crosslinking agent, a functionalization reagent and an initiator; And (b) contacting the organic phase with the aqueous phase under time and temperature conditions effective to form polymeric beads.

Oligomeric Synthesis, Oligonucleotide Synthesis, Solid Supports, Polymeric Beads

Description

POLYMERIC BEADS FOR OLIGOMER SYNTHESIS

FIELD OF THE INVENTION The present invention relates to solid support media for use in oligomer synthesis, methods of making the media, and methods of using the media.

Solid state synthesis is applicable to the preparation of a wide range of polymeric compounds such as nucleobase-containing polymers (eg oligonucleotides and analogs thereof), amino acid containing polymers (eg proteins, peptides, and analogs thereof). Solid state synthesis is also applicable to the preparation of compounds by combination methods.

Oligonucleotides have been used in a variety of biological and biochemical applications. It is used as primers and probes for polymerase chain reaction (PCR), as antisense agents used in target identification, drug discovery and development, as ribozymes, as aptamers, and as general stimulants of the immune system. come. As the popularity of oligonucleotides has increased, the demand for larger batches, and larger numbers of smaller batches, has gradually increased. In addition, emphasis has been placed on reducing the cost of oligonucleotide synthesis and on improving the purity of the oligonucleotide product and increasing the yield.

Many innovations have been introduced in the field of oligonucleotide synthesis. Among these innovations, excellent orthogonal protecting groups, activators, reagents and synthetic conditions have been developed. The oligonucleotides themselves have been variously modified and improved. These include chemistry that improves the affinity of oligonucleotides for specific targets, chemistry that increases the stability of oligonucleotides in vivo, and chemistry that enhances the pharmacokinetic (PK) and toxicological (Tox) properties of oligonucleotides. . Such new chemistries generally involve chemical modification of one or more constituent portions of the oligonucleotide.

Thus the term "oligonucleotide" includes naturally-occurring oligonucleotides, as well as a class of compounds in which modified oligonucleotides are included. Both naturally-occurring oligonucleotides and modified oligonucleotides have proven to be useful in a variety of circumstances and can all be prepared by similar processes with appropriate modifications made to account for the specific modifications employed. Naturally occurring oligonucleotides, ie, short strands of DNA or RNA, can be embodied as being members of the formulas designated oligo-RNA and oligo-DNA, respectively:

Wherein m is an integer from 1 to about 100 and Bx is a naturally occurring nucleobase.

Since phosphates dissociate easily at neutral pH, at physiological pH, oligonucleotides will occur as anions, and generally oligonucleotides will occur as amorphous salts in the amorphous or crystalline solid phase. Thus, unless otherwise modified, the term “oligonucleotide” includes each of the anionic, salt and free acid forms described above.

In essence, a naturally occurring oligonucleotide can be thought of as an oligomer of m monomeric subunits represented by the following nucleotides:

Wherein each Bx is a nucleobase and the last residue is a nucleoside (ie, a nucleotide without a 3'-phosphate group).

As mentioned above, various chemical modifications have been made to the oligonucleotides to improve the affinity, stability, PK, Tox, and other properties of the oligonucleotides. In general, the term oligonucleotide, as used in the art, specifically includes compounds of the formula:

[Wherein m is an integer from 1 to about 100, each G ₁ is O or S, each G ₂ is OH or SH, each G ₃ is O, S, CH ₂ , or NH, respectively G ₅ is a divalent moiety such as O, S, CH ₂ , CFH, CF ₂ , -CH = CH- and each R ₂ ′ is H, OH, O-rg (where rg is Or a 2′-substituent, or bridge with R ₄ ′, each R ₃ ′ is H, a substituent, or bridge with R ₄ ′, and each R ₄ ′ Is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′, each q is 0 or 1, and each R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′, each G ₆ is O, S, CH ₂ or NH, and each G ₇ is H, PO ₃ H ₂ , or a conjugate group , Each Bx is as described herein (ie, naturally occurring or modified) A base.

Standard methods for synthesizing oligonucleotides include the solid phase method described first by Carurthers et al. (See, eg, US Pat. No. 5,750,666, especially oligonucleotides, in particular phosphorothioate oligos, incorporated herein by reference). Starting materials and general methods of nucleotide preparation are disclosed and see column 3-58, a part of which is expressly incorporated herein by reference). This method was later improved by Koester et al. (See, eg, US Patent RE34,069, incorporated herein by reference, in particular the column which is disclosed and specifically incorporated herein by reference). . This method has been further improved by various inventors as discussed in more detail below. In particular, methods of RNA synthesis are disclosed in US Pat. Nos. 6,111,086, 6,008,400, and 5,889,136, each incorporated herein by reference in their entirety. Particularly relevant are columns 7-20 of US Pat. No. 6,008,400, which is expressly incorporated herein by reference.

A general process for preparing oligonucleosides by the method of Koester et al. Will be described below.

First, the synthetic support is prepared by covalently linking the appropriate nucleoside to the solid support medium (SS) via a linker. Such synthetic supports are as follows:

Wherein SS is a solid support medium and LL is a linking group connecting the nucleoside to the support via G ₃ . In general, the linking group covalently binds the final 3'-nucleoside (and thus oligonucleotide of the generator) to the solid support medium during synthesis, but is orthogonal to the conditions under which the 5'-protecting group and if applicable any 2'-protecting group are removed. Difunctional groups which are cleaved under phosphorus conditions. T 'is a removable protecting group and the remaining variables are defined above and described in more detail below. Suitable synthetic supports can be obtained under the trade name Primer Support 200 ™ from Amersham Biosciences. The solid support medium to which the synthetic support is attached is then swelled in a suitable solvent such as acetonitrile and one of the available solid phase synthetic instruments such as a synthesizer available from Amersham Biosciences such as AKTAoligopilot ™, or OligoProcess ™ brand DNA / It can be introduced into a column of an RNA synthesizer.

In this method, the synthesis is carried out from the 3'-end to the 5'-end of the oligomer. In each cycle, the following steps are performed: (1) removal of T ', (2) coupling, (3) oxidation, (4) capping. Each step (1)-(4) can be followed by one or more washing steps, generally followed by washing the soluble material out of the column, or pushing the reagents and / or activators through the column. To this, or for both, a cleaning solvent is introduced into the column. Steps (1)-(4) are depicted below:

In general, T 'is selected so that it can be removed under conditions orthogonal to those used to cleave oligonucleotides from the solid support medium at the end of the synthesis, as well as those used to remove other protecting groups used during synthesis. The protecting group recognized in the art for oligonucleotide synthesis is DMT (4,4'-dimethoxytrityl). DMT groups are particularly useful because they are removable under mild acid conditions. Thus, an acceptable removal reagent is 3% DCA in a suitable solvent such as acetonitrile. If used, the washing solvent may conveniently be acetonitrile.

Typically the support is a controlled pore glass or polymeric bead support. Some polymeric supports are disclosed in the following patents: US Pat. No. 6,016,895, each of which is expressly incorporated herein by reference; U.S. Patent 6,043,353; U.S. Patent 5,391,667 and U.S. Patent 6,300,486.

After removal of the protecting group T ', the next step in the synthesis cycle is the coupling of the next nucleoside synthon. This is accomplished by reacting the deprotected nucleoside bound to the support with nucleoside phosphoramidite in the presence of an activator, as shown below:

Amidite has the following structure:

[Wherein pg is a phosphorus protecting group such as cyanoethyl group and NR _N1 R _N2 is an amine leaving group such as diisopropyl amino]. For information on the preparation of amidite, see Koester et al., Supra. Typically used activators include, for example, tetrazole, dicyano imidazole, or pyridinium salts. Other suitable amidites, and methods of making amidites, are described in the following patents: U. S. Patent Nos. 6,133, 438, each of which is expressly incorporated herein by reference, in particular because it is particularly relevant to the manufacture of amidites; U.S. Patent 5,646,265; U.S. Patent 6,124,450; U.S. Patent 5,847,106; U.S. Patent 6,001,982; U.S. Patent 5,705,621; U.S. Patent 5,955,600; U.S. Patent 6,160,152; U.S. Patent 6,335,439; U.S. Patent 6,274,725; U.S. Patent 6,329,519. Suitable activators are described in the patents of Carurther et al. And in the patents of Koester et al. Particularly suitable activators are described in the following patents: US Pat. No. 6,031,092 and US Pat. No. 6,476,216, each of which is expressly incorporated herein by reference.

The next step in the synthesis cycle is oxidation, which indicates that the P (III) species are oxidized to the P (V) oxidation step with a suitable oxidant:

[Wherein, G ₁ is O or S].

The oxidant is an oxidizing agent suitable for the introduction of G ₁ . When G ₁ is oxygen, suitable oxidizing agents are described in the patents of Carurthers et al., Supra. If G ₂ is sulfur, the oxidant may also be referred to as a thiation agent or a sulfur-transfer reagent. Suitable thiatating agents include the so-called Beaucage reagent, 3H-1,2-benzothiol, phenylacetyl disulfide (also referred to as PADS; see, for example, US Pat. Nos. 6,114,519 and 6,242,591, each incorporated herein by reference). And thiouram disulfides (eg, N, N, N ', N'-tetramethylthiouram disulfides disclosed in US Pat. No. 5,166,387). The wash may be a suitable solvent such as acetonitrile.

An oxidation step is followed by a capping step, which is not illustrated herein but is an important step in the synthesis, as it allows the free 5'-OH groups that do not undergo the coupling step in step 1 to be blocked from coupling in subsequent synthesis cycles. to be. Suitable capping reagents are described in the patents of Carurthers et al., The patents of Koester et al., And other patents described herein. Suitable capping reagents include combinations of acetic anhydride and N-methylimidazole.

Synthesis cycle steps (1)-(4) are repeated n-1 (if desired) to produce support-binding oligonucleotides:

Wherein each variable is as defined herein.

In general, the protecting group pg may be removed by a method as described above in Carurthers et al. Or Koester et al. If pg is a cyanoethyl group, the method of Koester et al., for example, reaction with a basic solution, is generally suitable for the removal of phosphorus protecting groups. In some cases, it is desirable to avoid the formation of adducts such as N 1 -cyanoethyl thymidine groups. In such cases, preference is given to including tertiary amine reagents such as triethylamine (TEA) as taught in US Pat. No. 6,465,628, which is expressly incorporated herein by reference. In general, when a nucleobase is protected, it is deprotected under basic conditions. Deprotected oligonucleotides are cleaved from the support to provide the following 5′-protected oligonucleotides:

It can then be purified by reverse phase liquid chromatography, deprotected, desalted, lyophilized or otherwise dried at 5'-end in acetic acid and stored in an inert atmosphere until needed. Optionally, G ₃ H groups can be derivatized with conjugated groups. The resulting oligonucleotides can be visualized as having the formula:

Synthesis on solid phase support media is known, but in particular loading (in mmol, or simply mmol / g of the first nucleoside bound to solid support media per gram of solid support media), consistent swellability during various synthesis cycles With regard to the quality of the full length oligomers produced during the synthesis, and there is a need for solid support media with improved properties. In addition, the support should be easy to manufacture. The present invention relates to these and other important objects as well.

Summary of the Invention

In some embodiments, the present invention provides a method of making polymeric beads comprising providing an organic phase and contacting the organic phase with an aqueous phase under time, temperature and pressure conditions effective to form the polymeric beads.

In some embodiments, the organic phase comprises monomers, initiators and organic solvent (s) and the aqueous phase comprises water and dispersing reagents. In a preferred embodiment, the monomers include olefin monomers, crosslinking monomers and functionalized monomers. In another preferred embodiment, the organic solvent (s) are one or more liquid hydrocarbons; And / or alcohols having 5 to 12 carbon atoms. In another preferred embodiment of the invention, the olefin monomer comprises at least one aryl-vinyl compound and preferably comprises styrene and / or ethylstyrene. In another preferred embodiment, the crosslinking monomer is an olefinic crosslinking monomer having two non-conjugated vinyl groups, preferably attached to an aromatic moiety wherein the two non-conjugated vinyl groups are five or six membered aromatic rings, most preferred Preferably divinylbenzene. In another preferred embodiment, the functionalized monomer is an olefinic monomer having a protected functional group, or a protected hydroxyl group, which, when deprotected, yields a functional group capable of reacting with an acid or acid anhydride to form an ester or an amide. It is an olefin type monomer which has, Preferably it is acetoxy styrene. In another preferred embodiment, the initiator is a stabilized peroxide or azo compound, most preferably benzoylperoxide. In another preferred embodiment, the organic solvent is at least one liquid alkane, benzene, toluene, xylene and / or an alcohol having 5 to 12 carbon atoms, preferably the organic solvent is at least one octane, most preferably isooctane, And / or 2-ethylhexanol. In another preferred embodiment, the dispersing reagent comprises polyalcohol, preferably polyvinylalcohol.

In some embodiments of the invention, the various components are present in the following amounts: The weight percentage of the olefin monomer initially present in the monomer is from about 60% to about 96%; The weight percentage of crosslinking monomer initially present in the monomer is from about 3% to 9.9%; The weight percentage of the functionalized monomer initially present in the monomer is from about 1% to about 20%; The weight percentage of monomer initially present in the organic phase is from about 33% to about 67%; The weight percentage of organic solvent initially present in the organic phase is from about 33% to about 67%; The weight percentage of the liquid hydrocarbon present in the organic solvent is about 0% to about 80%; The weight percentage of the 5 to 12 carbon atoms initially present in the organic solvent is from about 20% to about 100%; The weight percentage of the dispersing reagent initially present in the aqueous phase is from about 0.01% to about 20%.

In another preferred embodiment, the various components are present in the following amounts: The weight percentage of the olefin monomer initially present in the monomer is about 75% to about 94%; The weight percentage of crosslinking monomer initially present in the monomer is about 4% to 9.9%; The weight percentage of the functionalized monomer initially present in the monomer is from about 2% to about 10%; The weight percentage of monomer initially present in the organic phase is from about 35% to about 60%; The weight percentage of organic solvent initially present in the organic phase is from about 40% to about 65%; The weight percentage of hydrocarbons present in the organic solvent is about 5% to about 70%; The weight percentage of alcohol having 5 to 12 carbon atoms initially present in the organic solvent is from about 30% to about 95%.

In another preferred embodiment, the various components are present in the following amounts: The weight percentage of the olefin monomer initially present in the monomer is from about 82% to about 91.5%; The weight percentage of crosslinking monomer initially present in the monomer is about 5.5% to 9.9%; The weight percentage of the functionalized monomer initially present in the monomer is from about 3% to about 8%; The weight percentage of monomer initially present in the organic phase is about 40% to about 50%; The weight percentage of organic solvent initially present in the organic phase is from about 50% to about 60%; The weight percentage of the liquid hydrocarbon present in the organic solvent is about 10% to about 60%; The weight percentage of alcohol having 5 to 12 carbon atoms initially present in the organic solvent is from about 40% to about 90%.

In another specific embodiment of the present invention, the step of contacting the organic phase with the aqueous phase is about 25 ° C. to about 95 ° C., more preferably about 70 ° C. to about 85 ° C., and most preferably about 75 ° C. to about Occurs at a temperature of 80 ° C.

The present invention is a method of synthesizing polynucleotides of a predetermined sequence, comprising using a polystyrene support prepared from a plurality of monomers comprising a crosslinking monomer, wherein about 3% by weight of the crosslinking monomer initially present in the plurality of monomers is present. Also provided is a method that is from 9.9% by weight. In some preferred embodiments, the crosslinking monomer initially present in the plurality of monomers is about 5.5% to 9.9%, most preferably about 7%. In another preferred embodiment, the crosslinking monomer is divinylbenzene.

The present invention also provides polymeric beads formed by any of the methods described herein. In some embodiments, the loading capacity of the beads is about 100 μmol per gram of beads to about 350 μmol per gram of beads. In some embodiments, the average particle size of the beads is about 5 to about 500 μm, preferably about 10 to about 300 μm, most preferably about 30 to about 150 μm. In some embodiments, the average pore size of the beads is about 5 to about 500 nm, preferably about 10 to about 100 nm, most preferably about 20 to about 100 nm. In some embodiments, the specific surface area of the beads is about 5 to about 200 m 2 / g, preferably about 10 to about 100 m 2 / g Most preferably about 20 to about 70 m 2 / g.

The present invention provides compounds of Formula I or Formula II:

[Meal:

G ₃ is O, S, CH ₂ , or NH;

G ₅ is O, S, CH ₂ , CFH, CF ₂ , or -CH = CH-;

G ₆ is O, S, CH ₂ , or NH;

Each R ₂ ′ is H, OH, O-rg (where rg is a removable protecting group), 2′-substituent, or bridges with R ₄ ′;

Each R ₃ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each R ₄ ′ is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′;

q is 0 or 1;

R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Bx is a nucleobase;

T 'is H or a removable protecting group;

LL is a linking moiety;

SS is the beads produced by the method described herein;

[Meal:

m is an integer from 0 to about 100;

Each G ₁ is O or S;

Each G ₂ is OH or SH;

Each G ₃ is O, S, CH ₂ , or NH;

Each G ₅ is O, S, CH ₂ , CFH, CF ₂ , or —CH═CH—;

Each R ₂ ′ is H, OH, O-rg (where rg is a removable protecting group), 2′-substituent, or bridges together with R ₄ ′;

Each R ₃ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each R ₄ ′ is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′;

Each q is 0 or 1;

Each R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each G ₆ is independently O, S, CH ₂ or NH;

Each Bx is a nucleobase;

pg is a removable phosphorus protecting group;

T 'is a removable protecting group;

LL is a linking moiety;

SS is beads produced by the methods described herein.

In another embodiment, the present invention provides a method for preparing an organic phase comprising (a) providing an organic phase comprising an olefin monomer, a crosslinking monomer, a functionalized monomer and an initiator; And (b) contacting the organic phase with the aqueous phase under time, temperature conditions effective for the polymeric beads to form.

In some embodiments, the aqueous phase comprises water and a dispersing reagent, and in some embodiments, the dispersing reagent comprises polyalcohols such as polyvinyl alcohol.

In some embodiments, the aqueous phase comprises water and a dispersing reagent, and the dispersing reagent comprises, in some embodiments, a polyalcohol; The organic phase comprises at least one liquid hydrocarbon and / or an organic solvent comprising an alcohol having 5 to 12 carbon atoms.

In some embodiments, the olefin monomers contain a single non-aromatic unsaturated group. In some additional embodiments, the olefin monomer is an aryl-vinyl compound, for example styrene.

In some embodiments, the crosslinking monomer is an olefinic crosslinking monomer. In some embodiments, the crosslinking monomer is an olefinic crosslinking monomer having two non-conjugated vinyl groups. In some further embodiments, the crosslinking monomer is an olefinic crosslinking monomer attached to an aromatic moiety wherein the two non-conjugated vinyl groups are five or six membered aromatic rings. In some embodiments, the crosslinking monomer is divinylbenzene.

In some embodiments, the functionalized monomer is an olefinic functionalized monomer having a protected functional group, and when the protected functional group is deprotected, a functional group capable of reacting with an acid or acid anhydride to form an ester or an amide, preferably Is calculated. In some embodiments, the functionalized monomer is an olefinic monomer having a protected hydroxyl group. In some embodiments, the functionalized monomer is at least one propanoyloxystyrene or acetoxystyrene, most preferred is acetoxystyrene.

In some embodiments, the organic phase further comprises an organic solvent, which in some embodiments comprises at least one liquid hydrocarbon and / or an alcohol having 5 to 12 carbon atoms. In some embodiments, the organic solvent is one or more liquid hydrocarbons, such as one or more liquid alkanes, benzene, toluene, xylene, for example octane, for example isooctane and / or alcohols having 5 to 12 carbon atoms, for example For example 2-ethylhexanol.

In some embodiments, the polymerization initiator is a stabilized peroxide, for example benzoylperoxide or azo compound.

In some embodiments, the olefin monomer is an aryl-vinyl compound and the crosslinking monomer is an olefinic crosslinking monomer having two non-conjugated vinyl groups. In a further embodiment, the olefin monomer is an aryl-vinyl compound and the crosslinking monomer is an olefinic crosslinking monomer attached to an aromatic moiety wherein two non-conjugated vinyl groups are five or six membered aromatic rings. In some additional embodiments, the olefin monomer is styrene and the crosslinking monomer is divinylbenzene. In some further embodiments, the olefin monomer is an aryl-vinyl compound, and the functionalized monomer has a protected functional group that, when deprotected, yields a functional group that can react with an acid or acid anhydride, preferably forming an ester or an amide. It is an olefin monomer.

In some embodiments, the olefin monomer is an aryl-vinyl compound and the functionalized monomer is an olefinic monomer having a protected hydroxyl group. In a further embodiment, the olefin monomer is styrene or ethylstyrene and the functionalized monomer is acetoxystyrene. In a further embodiment, the crosslinking monomer is an olefinic monomer attached to an aromatic moiety wherein the two non-conjugated vinyl groups are 5 or 6 membered aromatic rings, and the functionalized monomer reacts with the acid or acid anhydride when deprotected to give ester or It is an olefinic monomer having a protected functional group which yields a functional group capable of forming an amide. In a further embodiment, the crosslinking monomer is an olefinic crosslinking monomer attached to an aromatic moiety wherein two non-conjugated vinyl groups are 5 or 6 membered aromatic rings, and the functionalized monomer is an olefinic monomer having a protected hydroxyl group. . In a further embodiment, the crosslinking monomer is divinylbenzene and the functionalizing monomer is acetoxystyrene.

In some embodiments, the olefin monomer is an aryl-vinyl compound, the crosslinking monomer is an olefinic crosslinking monomer attached to an aromatic moiety wherein two non-conjugated vinyl groups are five or six membered aromatic rings, and the functionalized monomer is deprotected. An olefinic monomer having a protected functional group which, when reacted with an acid or acid anhydride, yields a functional group capable of forming an ester or an amide.

In a further embodiment, the olefin monomer is an aryl-vinyl compound, the crosslinking monomer is an olefinic crosslinking monomer attached to an aromatic moiety wherein two non-conjugated vinyl groups are five or six membered aromatic rings, and the functionalized monomer is a protected hydride. It is an olefin type monomer which has a hydroxyl group.

In a further embodiment, the olefin monomer is styrene, the crosslinking monomer is divinylbenzene and the functionalized monomer is acetoxystyrene.

In some of each of the above embodiments, the aqueous phase is water and a dispersing reagent, and in some embodiments, the dispersing reagent comprises polyvinylalcohol.

In some embodiments of the method, the contacting further comprises shaking the aqueous and organic phases, for example by stirring the aqueous and organic phases. In a further embodiment, the method further comprises washing the beads. In some embodiments, the beads are washed with one or more wash solvents and at least one of the wash solvents includes acetone, water or methanol.

In some embodiments of the method, the divinylbenzene may comprise from about 3 to about 20 weight percent of total monomers initially present in the organic phase; Or about 4 to about 15 weight percent of total monomers initially present in the organic phase; Or initially in an organic phase in an amount that is about 5.5 to about 10 weight percent of the total monomers present in the organic phase.

In some embodiments of the method, the acetoxystyrene comprises from about 1 to about 20 weight percent of total monomers initially present in the organic phase; Or about 2 to about 10 weight percent of total monomers initially present in the organic phase; Or initially present in the organic phase in an amount that is about 3 to about 8 weight percent of the total monomers present in the organic phase.

In some embodiments in which the aqueous phase comprises water and a dispersing reagent, the organic phase further comprises an organic solvent comprising at least one liquid hydrocarbon and / or an alcohol having 5 to 12 carbon atoms. In some such embodiments, the weight percentage of organic solvent initially present in the organic phase is about 33% to about 67%; The weight percentage of total monomers initially present in the organic phase is from about 33% to about 67%; Weight percentage of the olefin monomer initially present in the total monomers is from about 60% to about 96%; The weight percentage of crosslinking monomer initially present in the monomer is from about 3% to about 20%; The weight percentage of the functionalized monomer initially present in the monomer is from about 1% to about 20%; The weight percentage of hydrocarbons present in the organic solvent is about 0% to about 80%; The weight percentage of alcohol having 5 to 12 carbon atoms initially present in the organic solvent is from about 20% to about 100%. In further such embodiments, the weight percentage of organic solvent initially present in the organic phase is from about 40% to about 65%; The weight percentage of total monomers initially present in the organic phase is from about 35% to about 60%; The weight percentage of the olefin monomer initially present in the monomer is from about 75% to about 94%; The weight percentage of crosslinking monomer initially present in the total monomer is about 4% to about 15%; The weight percentage of functionalized monomer initially present in the total monomer is from about 3% to about 8%; The weight percentage of hydrocarbons present in the organic solvent is about 5% to about 70%; The weight percentage of alcohol having 5 to 12 carbon atoms initially present in the organic solvent is from about 30% to about 95%. In further such embodiments, the weight percentage of organic solvent initially present in the organic phase is from about 50% to about 60%; The weight percentage of total monomers initially present in the organic phase is from about 40% to about 50%; The weight percentage of olefin monomer initially present in the total monomer is from about 82% to about 91.5%; The weight percentage of crosslinking monomer initially present in the total monomers is from about 5.5% to about 10%; The weight percentage of the functionalized monomer initially present in the monomer is from about 3% to about 8%; The weight percentage of hydrocarbons present in the organic solvent is about 10% to about 60%; The weight percentage of alcohol having 5 to 12 carbon atoms initially present in the organic solvent is from about 40% to about 90%. In some such embodiments, the dispersing reagent comprises polyvinyl alcohol. In such further embodiments, the olefin monomer is styrene or ethylstyrene, the crosslinking monomer is divinylbenzene, the functionalized monomer is acetoxystyrene, the hydrocarbon is isooctane and the alcohol having 5 to 12 carbon atoms is 2-ethylhexanol. . In some such embodiments, the weight percentage of the dispersing reagent initially present in the aqueous phase is about 0.01% to about 20%.

In some embodiments of the methods described herein, the organic and aqueous phases are from about 25 ° C. to about 95 ° C .; Or about 40 ° C. to about 90 ° C .; Or about 70 ° C. to about 85 ° C .; Or heated to a temperature of about 75 ° C to about 80 ° C.

The present invention comprises the steps of (a) providing polymeric beads produced by the method of the present invention and optionally reacting said beads with an activating reagent to provide activated polymeric beads; (b) reacting said optionally activated polymeric beads with one or more linking reagents to produce beads having a support-binding linker; And (c) linking the support-binding linker with the nucleoside to form a support-binding nucleoside.

The present invention comprises the steps of (a) providing polymeric beads produced by the method of the present invention and optionally reacting said beads with an activating reagent to provide activated polymeric beads; And (b) reacting said optionally activated polymeric beads with a linker-containing nucleoside to form a support-binding nucleoside.

The present invention provides (a) a support-binding nucleoside prepared by any of the above methods of the present invention as described herein, wherein the support-binding nucleoside has at least one protected hydroxyl group. step; (b) deprotecting the hydroxyl group of the support-binding nucleoside; (c) contacting the support-binding nucleoside with an activated protected nucleoside to produce a phosphite intermediate; (d) contacting the phosphite intermediate with an oxidizing reagent to produce the phosphortriester intermediate; (e) optionally capping unreacted nucleosides; (f) optionally repeating steps (b)-(e) one or more times; And (g) cleaving the oligonucleotide from the solid support. In some such embodiments, the deprotection step of step (b) comprises the removal of an acid-labile protecting group.

The present invention provides (a) a support-binding nucleoside prepared by any of the above methods of the present invention as described herein, wherein the support-binding nucleoside has at least one protected hydroxyl group. step; (b) deprotecting the hydroxyl group of the support-binding nucleoside; (c) contacting the support-binding nucleoside with an activated protected nucleoside to produce a phosphite intermediate; (d) contacting the phosphite intermediate with an oxidizing reagent to produce the phosphortriester intermediate; (e) optionally capping unreacted nucleosides; (f) optionally repeating steps (b)-(e) one or more times; And (g) cleaving the oligonucleotide from the solid support. In some such embodiments, the deprotection step of step (b) comprises the removal of acid-degradable protecting groups.

The present invention also provides a compound of formula (I)

(I)

[Wherein LL is a linking moiety; SS is a bead of the invention as described herein; Other component variables are as described below]. In some embodiments, G ₃ , G ₅ and G ₆ are each O; In further embodiments, one of R ₂ ′ is H and R ₃ ′ and R ₄ ′ are each H.

The invention further provides compounds of formula II:

≪ RTI ID = 0.0 &

Wherein the component variables are as described below. In some embodiments, each G ₃ , G ₅ and G ₆ is O. In further such embodiments, one of each adjacent R ₂ 'group pair is H and each R ₃ ′ and each R ₄ ′ is H.

The present invention provides a process for preparing a compound of Formula III comprising the steps of:

And b) reacting the compound of Formula III with the beads of the present invention as described herein under conditions effective to form the compound of Formula I. In some embodiments, G ₃ , G ₅ and G ₆ are each O. In further embodiments, one of R ₂ ′ is H and R ₃ ′ and R ₄ ′ are each H.

The present invention comprises the steps of: a) providing a bead of the invention as described herein; b) attaching one end of the bifunctional linker LL to the functional group of the bead; And c) reacting the other end of the bifunctional linker LL with a compound of formula IV to form a compound of formula I:

In some embodiments, G ₃ , G ₅ and G ₆ are each O. In further embodiments, one of R ₂ ′ is H and R ₃ ′ and R ₄ ′ are each H.

The present invention provides a process for the preparation of a compound of formula II:

≪ RTI ID = 0.0 &

Wherein LL is a linking moiety, SS is a bead of the invention as described herein and other constituent variables are as described below], a) providing a compound of formula I as described above Making;

(I)

Wherein T 'is a removable protecting group; b) removing the protecting group T ′ to form a deprotected support-binding nucleoside; c) the deprotected support-binding nucleoside of step (b) is reacted with a protected nucleoside amidite of Formula V:

Wherein the component variables are as defined below, forming a phosphite compound of formula VI:

d) oxidizing or sulfiding a phosphate of formula VI to form a compound of formula II wherein m is 1; e) optionally capping unreacted nucleosides; And f) optionally repeating steps (b)-(e) one or more times. In further embodiments, one of each adjacent R ₂ ′ group pair is H and each R ₃ ′ and each R ₄ ′ is H.

In some embodiments of the above synthetic method, the method further comprises cleaving the resulting oligonucleotide from the solid support.

The present invention is directed to formula (a) of formula SS-LL-ZZ, wherein SS is the polymeric bead of the present invention, LL is any linker, and ZZ is capable of forming anchoring linkages with carboxyl or amino groups of amino acids. Providing a polymer substrate; (b) coupling the first amino acid synthesis unit having a protecting group to a carboxyl or amino group with a polymer substrate; (c) removing the protecting group from the first amino acid coupled to produce a free amino or carboxyl group; And (d) reacting the free amino or carboxyl group with a second amino acid synthetase to form a peptide chain, the method of producing an oligomer comprising an amino acid monomer. In some embodiments, (e) removing the protecting group from the second amino acid synthetase to produce a free amino or carboxyl group on the peptide chain; And (f) reacting the free amino or carboxyl group on the peptide chain with an additional protected amino acid synthetase to extend the peptide chain. In further embodiments, steps e and f are performed several times. Some embodiments further comprise cleaving said anchor linkage without substantially breaking said peptide chain. In some embodiments, amino acid syntheses are naturally occurring amino acids. In another embodiment, the amino acid synthesis unit is a peptide nucleic acid synthesis unit.

The present invention provides a process for the preparation of a compound of formula X:

Wherein the constituent parameters are as defined below: (a) Formula SS-LL-ZZ, wherein SS is the polymeric bead of claim 67, LL is a linker, and ZZ forms a fixed linkage with an amino acid Providing a polymer substrate; (b) coupling the first amino acid of formula XX to the polymer substrate via the anchoring linkage:

Wherein the component variables are as defined below; (c) removing said amino protecting group from the first amino acid coupled to produce a free amino group; And (d) reacting the free amino group with a second amino acid of Formula (XX) to form a peptide chain. In some embodiments, (e) removing said amino protecting group from said second amino acid to produce a terminal free amino group amino group on said peptide chain; And (f) reacting said free amino group on said peptide chain with an additional amino acid of formula (XX) to extend said peptide chain. In some embodiments, steps e and f are performed several times. Some embodiments further comprise removing one or more protecting groups remaining on the amino acid moiety of the peptide chain. Some embodiments further comprise cleaving said anchor linkage without substantially breaking said peptide chain. In some embodiments, the chemical groups capable of forming a fixed linkage are chloro-, bromo- and iodo-substituted alkyl, amino-substituted alkyl, amino and aryl-substituted alkyl, amino- and alkylaryl-substituted alkyl, Hydroxy-substituted alkyls, or derivatives thereof having spacer groups that can be cleaved substantially without degradation of the polypeptide. In some embodiments, chloro-substituted alkyl is chloromethyl, amino-substituted alkyl is aminomethyl, amino- and alkyl-substituted aryl is "-aminobenzyl, and amino- and alkylaryl-substituted alkyl is" -amino- And hydroxy-substituted alkyl is hydroxymethyl. In some embodiments, the chemical group is amino-substituted alkyl, amino- and aryl substituted alkyl, and Derived from an amino-containing moiety selected from amino- and alkylaryl-substituted alkyl; the chemical group is 4- (haloalkyl) aryl-lower alkanoic acid, Boc-aminoacyl-4- (oxymethyl) aryl-lower alkanoic acid, N-Boc-p-acylbenzhydrylamine, N-Boc-4 '-(lower alkyl) -p-acylbenzhydrylamine, N-Boc-4'-(lower alkoxy) -p-acylbenzhydryl Amines, and spacer groups derived from the group consisting of 4-hydroxymethylphenoxy-lower alkanoic acid .

In some embodiments, compound X has the formula:

Wherein the component variables are as defined below.

In further embodiments, Compound X has the formula:

Wherein the component variables are as defined below.

In some embodiments, amino acids of Formula XX have the formula:

Wherein the component variables are as defined below.

In further embodiments, the amino acid of formula XX has the formula:

Wherein the component variables are as defined below.

In some embodiments, the loading dose of polymeric beads of the present invention is at least about 50 μmol per gram of beads; At least about 100 μmol per gram of beads; At least about 150 μmol per gram of beads; At least about 200 μmol per gram of beads; At least about 250 μmol per gram of beads; At least about 300 μmol per gram of beads; At least about 350 μmol per gram of beads; At least about 400 μmol per gram of beads; Or at least about 450 μmol per gram of beads. In some embodiments, the loading capacity of the beads is about 100 μmol per gram of beads to about 350 μmol per gram of beads.

Advantageously, the beads of the invention are relatively easy to manufacture and use with various linkers and nucleosides. In addition, the beads of the invention can be used in a variety of automated solid-phase synthesis instruments. The beads of the invention are excellent in loading capacity and may contain more than 100 mmol of the first nucleoside / oligonucleotide per gram of support. The beads of the present invention are also compatible with a variety of synthetic reagents and solvents, constant swelling properties over a wide range of synthetic solutions, advantageous backpressure profiles, good uniformity of bead and pore sizes, and resulting good synthesis (full length oligomers) Including performance, it has excellent physical properties.

The bead manufacturing method of the present invention can be easily implemented, is scalable, and has a good cost profile. In addition to the advantages associated with ease of manufacture and use as described above, the beads of the invention can be applied to the use of various linkers that can be used to covalently attach nucleosides, amino acids, or derivatives thereof to a support. . In addition, the beads of the present invention allow for efficient production of high quality (full length) oligonucleotides and other polymeric species.

The present invention provides crosslinked functionalized polymeric beads for oligomer synthesis, methods for their preparation, methods for using them in oligomer synthesis, support-bound nucleosides, and methods for making and using them, as well as support-bound oligonucleotides and amides. -Linked oligomers, methods of use thereof, and oligonucleotides and amide-linked oligomers prepared using polymeric beads according to the present invention.

In some embodiments, the beads comprise (a) providing an organic phase comprising an olefin monomer, a crosslinking monomer, a functionalized monomer and an initiator; And (b) contacting the organic phase with the aqueous phase under time, temperature conditions effective to form polymeric beads.

In some embodiments, the beads of the invention comprise crosslinked polyolefins. In some preferred embodiments, the beads of the invention comprise crosslinked polystyrene. According to the process of the invention described herein, the beads are formed by free-radical reaction of a composition comprising an olefin and a crosslinking agent under conditions suitable for forming the beads having the desired properties. Preferred olefins are styrene.

The aqueous phase generally includes water and dispersing reagents, which are believed to promote the formation of stable beads. The dispersing reagent may be a single compound or a mixture of dispersants. In some embodiments, the dispersing reagent comprises one or more polyalcohols, polyacrylic acid, carboxymethyl cellulose, gelatin, calcium carbide, calcium phosphate, calcium sulfate, barium sulfate, bentonite. In some preferred embodiments, the dispersing reagent comprises or consists of polyvinyl alcohol. In some embodiments, the dispersing reagent is present in the aqueous phase in an amount from about 0.01% to about 20%, or from about 0.1% to about 10% by weight of the aqueous phase.

The organic phase includes olefin monomers, crosslinking monomers, functionalized monomers, and initiators. In some embodiments, the organic phase further comprises one or more organic solvents.

In some embodiments, the olefin monomers contain a single non-aromatic unsaturated group, such as a vinyl group. Preferably, the olefinic monomers also contain aryl moieties, for example phenyl groups. Thus, in some preferred embodiments, the olefin monomer is an aryl-vinyl compound. In a particularly preferred embodiment, the olefin monomer is styrene or ethyl styrene.

The organic phase comprises at least one crosslinking monomer, ie a bifunctional compound which crosslinks the polymerized chain. In some embodiments, the crosslinking monomer is a bis-olefinic crosslinking monomer; That is, molecules that contain two or more carbon-carbon double bonds that can form separate bonds. In some embodiments, the olefinic crosslinking monomer has two non-conjugated vinyl groups. In some embodiments, the olefinic crosslinking monomer has two non-conjugated vinyl groups attached to the aromatic moiety, and in some embodiments, the aromatic moiety is a five or six membered aromatic ring, for example a phenyl ring. In one preferred embodiment, the crosslinking monomer is 1,2-, 1,3-, or 1,4-divinylbenzene, or a mixture of one or more of these. More preferably, the crosslinking monomer comprises or consists of 1,3- or 1,4-divinylbenzene.

Beads of the invention further comprise one or more types of functional groups contributed by one or more functionalized monomers. The functionalizing monomer is a polymer or preferably monomer which contributes a functional group capable of forming a covalent bond to the linker moiety or directly to the functional group of the nucleoside, amino acid, or core moiety for combination synthesis. In a particularly preferred embodiment, the functional group is -OH. Functional groups may be contributed by functionalized monomers that are reactive with styrene and / or crosslinking monomers during polymerization. In some embodiments, the functionalized monomer has at least one olefinic group covalently attached to the functional group. In some preferred embodiments, the functionalized group is an acyloxyl group.

In a particularly preferred embodiment, the functionalizing agent has the formula:

Wherein R _F is selected from substituted or unsubstituted C ₁ -C ₁₀ alkyl.

Particularly preferred functionalized monomers are acetoxystyrene, propanoyloxystyrene, and acetoxystyrene is most particularly preferred.

In some embodiments, the functional group is protected with one or more protecting groups, which must be removed before oligomer synthesis. Such functional groups include acyl groups such as acetyl, propanoyl, benzoyl and other functional group protecting groups. Accordingly, embodiments of the present invention include both protected functionalized crosslinked polyolefinic polymers and deprotected functionalized crosslinked polyolefinic polymers. In some embodiments, when the functional group is deprotected, a functional group suitable for reaction with an acid or acid anhydride is produced, preferably an ester or amide is formed.

The organic phase may further comprise one or more organic solvents. Suitable solvents include one or more liquid hydrocarbons and / or alcohols of 5 to 12 carbon atoms. In some embodiments, the organic solvent comprises one or more liquid hydrocarbons, preferably one or more liquid alkanes, benzene, toluene, xylenes, for example one or more octanes and / or alcohols having 5 to 12 carbon atoms. In a preferred embodiment, the organic solvent comprises isooctane and 2-ethylhexanol.

The organic phase may further comprise one or more initiators. Suitable initiators include compounds capable of initiating free radical polymerization reactions, for example stabilized peroxides or azo compounds. In some preferred embodiments, the initiator is benzoyl peroxide, dilauroyl peroxide, 1,1-di (t-butylperoxy) -2-methylcyclohexane, di-t-butyl peroxide, distearoyl Peroxide, di-t-hexyl peroxide, t-butyl cumyl peroxide, 1,1-di (t-hexyl peroxy) -3,3,5-trimethylcyclohexane, 1,1-di (t-butyl Peroxy) cyclohexane, 1,1,3,3-tetramethylbutylperoxy-2-ethylhexanoate, t-hexyl peroxy-2-ethylhexanoate, 2,2'-azobisisobutyro Nitrile, 2,2'-azobis-2-methylbutyronitrile, or 2,2'-azobis-2,4-dimethylvaleronitrile. In one preferred embodiment, the initiator comprises or consists of benzoylperoxide.

In some preferred embodiments, the olefin monomer is styrene or ethyl styrene, the crosslinking monomer is divinylbenzene and the functionalized monomer is acetoxystyrene. In some such embodiments, the organic solvent is isooctane and 2-ethylhexanol and the initiator is benzoylperoxide.

In one preferred embodiment according to the invention, a process for preparing functionalized crosslinked polystyrene beads, suitable for oligonucleotide synthesis, comprising the steps of: (a) mixing water and polyvinyl alcohol (PVA) to form an aqueous solution; (b) mixing isooctane and a 2-ethylhexanol solvent, a styrene monomer, a divinylbenzene crosslinking monomer, a benzoylperoxide initiator and an acetoxystyrene functionalized monomer to form an organic solution; And (c) mixing the aqueous solution with the organic solution to form functionalized crosslinked polystyrene beads suitable for oligonucleotide synthesis.

According to some embodiments of the process of the invention, contacting the organic phase with the aqueous phase comprises shaking the two phases, for example by stirring. In some embodiments, the two phases are shaken for 5 hours, 7 hours, 10 hours, 12 hours, or 15 hours or more. In general, it is advantageous to stir the mixture of organic and aqueous phases at a fixed rate in order to facilitate the production of the desired bead size distribution. In some preferred embodiments, the mixture is stirred at 250 rpm for a desired time by paddles, anchor paddles, propellers or disk turbines.

Generally, the organic and aqueous phases are subjected to about 25 ° C. to about 95 ° C. during shaking; About 40 ° C. to about 90 ° C .; About 70 ° C. to about 85 ° C .; Or heated to a temperature of about 75 ° C to about 80 ° C.

After the reaction of the organic and aqueous phases, the beads are preferably washed with one or more washing solvents. In some embodiments, at least one of the washing solvents is acetone, water or methanol. Washing can be accomplished by any of several techniques for washing solid support media, for example by filtering the reaction mixture and washing the beads on the filter with a wash solvent. In some embodiments, the beads can then be separated by size, for example by dispersing the beads in a suitable solvent, such as acetone, and sieving the suspension to collect beads of the desired size. The beads can then be dried, for example under vacuum.

In some preferred embodiments, the crosslinking monomer, such as divinylbenzene, is about 3% to about 20%, about 4% to about 15%, or about 5.5% by weight of the total monomers initially present in the organic phase. It is initially present in the organic phase in an amount of from about 10% by weight.

In a further preferred embodiment, the molar ratio of crosslinking monomers such as divinylbenzene to the total monomers initially present in the organic phase is from about 1:27 to about 1: 8; From about 1:25 to about 1:10; Or about 1:20 to about 1:13.

In some further preferred embodiments, the functionalized monomer such as acetoxystyrene is about 1% to about 20%, about 2% to about 10%, or about 1% by weight of the total monomers initially present in the organic phase. It is initially present in the organic phase in an amount of from 3% to about 8% by weight.

In some further preferred embodiments, the molar ratio of functionalized monomers such as acetoxystyrene to monomers initially present in the organic phase is from about 1:99 to about 1:10; Or about 1:62 to about 1:18. In some further preferred embodiments, the molar ratio is about 1:46 to about 1:21.

In some preferred embodiments, the organic phase further comprises an organic solvent comprising at least one liquid hydrocarbon and / or an alcohol having 5 to 12 carbon atoms, preferably isooctane and 2-ethylhexanol;

The weight percentage of organic solvent initially present in the organic phase is about 33% to about 67%, or about 40% to about 65%, or about 50% to about 60%;

The weight percentage of total monomers initially present in the organic phase is about 33% to about 67%, or about 35% to about 60%, or about 40% to about 50%;

The weight percentage of the olefin monomer, preferably styrene or ethylstyrene, initially present in the total monomer is about 60% to about 96%, or about 75% to about 94%, or about 82% to about 91.5%;

The weight percentage of the crosslinking monomer, preferably at least one divinylbenzene, initially present in the total monomer is about 3% to about 20%, or about 4% to about 15%, or about 5.5% to about 10%;

The weight percentage of the functionalized monomer, preferably acetoxystyrene, initially present in the total monomer is about 1% to about 20%, or about 2% to about 10%, or about 3% to about 8%;

The weight percentage of hydrocarbons, preferably isooctane, present in the organic solvent is about 0% to about 80%, or about 5% to about 70%, or about 10% to about 60%;

The weight percentage of an alcohol having 5 to 12 carbon atoms, preferably 2-ethylhexanol, initially present in the organic solvent, is from about 20% to about 100%, or from about 30% to about 95%, or from about 40% to about 90%.

In some such embodiments, the aqueous phase comprises water and a dispersing reagent, preferably polyalcohol such as polyvinyl alcohol, wherein the dispersing reagent is from about 0.01% to about 20%, or from about 0.1% to about 10 of the weight of the aqueous phase. It is present in the aqueous phase in an amount which is%.

In some embodiments of the methods herein, the ratio of the volume of the aqueous phase to the volume of the organic phase is from about 2: 3 to about 50: 1; Or about 1: 1 to about 20: 1; Or about 3: 2 to about 10: 1.

The present invention comprises the steps of (a) providing polymeric beads produced by the method of the present invention and optionally reacting said beads with an activating reagent to provide activated polymeric beads; (b) reacting the activated polymeric beads with one or more linking reagents to produce beads having a support-linked linker; And (c) linking the support-binding linker with the nucleoside to form a support-binding nucleoside.

In a further embodiment, the present invention provides a method for preparing a polymeric bead comprising (a) providing polymeric beads produced by the method of the present invention and optionally reacting said beads with an activating reagent to provide activated polymeric beads; And (b) linking the activated polymeric beads with a linker-containing nucleoside to form a support-binding nucleoside.

In a further embodiment, the present invention provides a method of preparing a pharmaceutical composition comprising (a) providing a support-binding nucleoside prepared by the method of the invention; (b) deprotecting the hydroxy group of the support-binding nucleoside, for example by removing the acid-degradable protecting group; (c) contacting the support-binding nucleoside with an activated protected nucleoside to produce a phosphite intermediate; (d) contacting the phosphite intermediate with an oxidizing reagent to produce a phosphortriester intermediate; (e) optionally capping unreacted nucleosides; (f) optionally repeating steps (b)-(e) one or more times; And (g) cleaving the oligonucleotide from the solid support.

In some embodiments, the present invention provides a compound of Formula I:

(I)

[Meal:

G ₃ is O, S, CH ₂ , or NH;

G ₅ is O, S, CH ₂ , CFH, CF ₂ , or -CH = CH-;

G ₆ is O, S, CH ₂ , or NH;

Each R ₂ ′ is H, OH, O-rg (where rg is a removable protecting group), 2′-substituent, or bridges with R ₄ ′;

Each R ₃ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each R ₄ ′ is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′;

q is 0 or 1;

R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Bx is a nucleobase;

T 'is H or a removable protecting group;

LL is a linking moiety or a single bond;

SS is a bead of the invention as described herein].

In some preferred embodiments of the compound of Formula (I), G ₃ , G ₅ and G ₆ are each O. In some further preferred embodiments of the compound of Formula (I), at least one R ₂ ′ is H and R ₃ ′ and R ₄ ′ are each H.

According to the invention there is also provided a compound of formula II:

≪ RTI ID = 0.0 &

[Meal:

m is an integer from 0 to about 100;

Each G ₁ is O or S;

Each G ₂ is OH or SH;

Each G ₃ is O, S, CH ₂ , or NH;

Each G ₅ is O, S, CH ₂ , CFH, CF ₂ , or —CH═CH—;

Each R ₂ ′ is H, OH, O-rg (where rg is a removable protecting group), 2′-substituent, or bridges together with R ₄ ′;

Each R ₃ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each R ₄ ′ is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′;

Each q is 0 or 1;

Each R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each G ₆ is independently O, S, CH ₂ or NH;

Each Bx is a nucleobase;

T 'is a removable protecting group;

LL is a linking moiety;

SS is a bead of the invention as described herein].

In some preferred embodiments of the compound of Formula II, each G ₃ , G ₅ and G ₆ is O. In some further preferred embodiments of the compound of Formula II, at least one of each adjacent R ₂ ′ group pair is H, and each R ₃ ′ and each R ₄ ′ is H.

In a further embodiment, the present invention provides a process for the preparation of a compound of formula (I):

(I)

[Meal:

G ₃ is O, S, CH ₂ , or NH;

G ₅ is O, S, CH ₂ , CFH, CF ₂ , or -CH = CH-;

Each R ₂ ′ is H, OH, O-rg (where rg is a removable protecting group), 2′-substituent, or bridges with R ₄ ′;

Each R ₃ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each R ₄ ′ is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′;

q is 0 or 1;

R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Bx is a nucleobase;

pg is a removable phosphorus protecting group;

T 'is H or a removable protecting group;

LL is a linking moiety or a single bond;

SS is a bead of the invention as described herein],

a) providing a compound of Formula III:

[Formula III]

; And b) reacting the compound of Formula III with the beads of the present invention under conditions effective to form the compound of Formula I. In some embodiments of this method, G ₃ , G ₅ and G ₆ are each O. In some additional embodiments, at least one R ₂ ′ is H and R ₃ ′ and R ₄ ′ are each H.

In a further embodiment, the present invention provides a process for the preparation of a compound of formula (I):

(I)

[Meal:

G ₃ is O, S, or NH;

G ₅ is O, S, CH ₂ , CFH, CF ₂ , or -CH = CH-;

Each R ₂ ′ is H, OH, O-rg (where rg is a removable protecting group), 2′-substituent, or bridges with R ₄ ′;

Each R ₃ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each R ₄ ′ is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′;

q is 0 or 1;

R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Bx is a nucleobase;

T 'is H or a removable protecting group;

LL is a linking moiety;

SS is a bead of the invention as described herein],

a) providing a bead of the invention;

b) attaching one end of the bifunctional linker LL to the functional group of the bead; And

c) reacting the other end of the bifunctional linker LL with a compound of formula IV:

(IV)

There is also provided a method comprising the step of forming a compound of formula (I). In some embodiments, G ₃ , G ₅ and G ₆ are each O. In further embodiments, at least one R ₂ ′ is H and R ₃ ′ and R ₄ ′ are each H.

According to the invention, a process for the preparation of the compound of formula II:

≪ RTI ID = 0.0 &

[Meal:

m is an integer from 0 to about 100;

Each G ₁ is O or S;

Each G ₂ is OH or SH;

Each G ₃ is O, S, CH ₂ , or NH;

Each G ₅ is O, S, CH ₂ , CFH, CF ₂ , or —CH═CH—;

Each R ₂ ′ is H, OH, O-rg (where rg is a removable protecting group), 2′-substituent, or bridges together with R ₄ ′;

Each R ₃ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each R ₄ ′ is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′;

Each q is 0 or 1;

Each R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′;

Each G ₆ is O, S, CH ₂ or NH;

Each Bx is a nucleobase;

T 'is H or a protecting group;

LL is a linking moiety or a single bond;

SS is a bead of the invention as described herein],

a) providing a compound of formula (I);

(I)

Wherein T 'is a removable protecting group;

b) removing the protecting group T ′ to form a deprotected support-binding nucleoside;

c) the deprotected support-binding nucleoside of step (b) is reacted with a protected nucleoside amidite of Formula V:

(V)

Wherein R _N1 and R _N2 are alkyl having 1 to 6 carbon atoms, forming a phosphite compound of formula VI:

(VI)

d) oxidizing or sulfiding a phosphate of formula VI to form a compound of formula II wherein m is 1;

e) optionally capping unreacted nucleosides; And

f) Optionally, there is also provided a method comprising the step of repeating steps (b)-(e) one or more times.

In some embodiments, each G ₃ , G ₅ and G ₆ is O. In further embodiments, at least one of each adjacent R ₂ 'group pair is H and each R ₃ ′ and each R ₄ ′ is H. In some additional embodiments, the method further comprises cleaving the resulting oligonucleotide from the solid support.

The polymeric beads of the present invention are also useful for the preparation of amide-linked oligomers such as proteins, peptides, and analogs thereof, and amide-linked nucleobase- or nucleobase analog-containing oligomers such as peptide nucleic acids. The term "amino acid" as used herein refers to a monomeric species having both amino and carboxyl groups. Thus, "amino acids" include naturally occurring α-amino acids as seen in peptides and proteins, analogs thereof, other naturally occurring amino acids such as γ-amino butyric acid, and other non-naturally occurring amino acids from which oligomeric compounds can be prepared. Included. Thus, in some embodiments, the present invention provides a method of preparing an oligomer comprising an amino acid monomer,

(a) a polymer of the formula SS-LL-ZZ, wherein SS is the polymeric bead of the invention, LL is any linker, and ZZ is a chemical group capable of forming a fixed link with an carboxyl or amino group of an amino acid Providing a substrate;

(b) coupling a first amino acid synthetic unit with the polymer substrate via a carboxyl or amino group of an amino acid, wherein the synthetic unit has a protecting group on an uncoupled carboxyl or amino group;

(c) removing the protecting group from the first amino acid coupled to produce a free amino or carboxyl group; And

(d) reacting a free amino or carboxyl group with a second amino acid synthetase to form a peptide chain. In some embodiments:

(e) removing the protecting group from the second amino acid synthetase to produce a terminal free amino or carboxyl group on the peptide chain; And

(f) further comprising the step of extending said peptide chain by reacting said free amino or carboxyl group on said peptide chain with an additional protected amino acid synthetase.

In some embodiments where longer species are desired, steps e and f are performed several times. Once the desired sequence is achieved, the fixed linkage is cleaved without substantially breaking down the assembled chain.

In some preferred embodiments, the amino acid syntheses are naturally occurring amino acids, analogs thereof, or peptide nucleic acids.

In some additional embodiments, the present invention provides a method of preparing a compound of Formula X:

(X)

[Meal:

n is 2 or more,

Each L ¹ -L ⁿ is hydrogen, hydroxy, (C ₁ -C ₄ ) alkanoyl, naturally occurring nucleobase, non-naturally occurring nucleobase, aromatic moiety, DNA intercalator, nucleobase- Independently selected from the group consisting of a bonding group, a heterocyclic moiety, and a reporter ligand, ^{wherein at} least one of L ¹ -L ⁿ is a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercal Or a nucleobase-bonding group;

Each C ¹ -C ⁿ is (CR ⁶ R ⁷ ) _y , wherein R ⁶ is hydrogen and R ⁷ is selected from the group consisting of side chains of naturally occurring alpha amino acids, or R ⁶ and R ⁷ are hydrogen, ( C ₂ -C ₆ ) alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C ₁ -C ₆ ) alkoxy, (C ₁ -C ₆ ) alkylthio, NR ³ R ⁴ and SR ⁵ (where R ³ and R ⁴ is as defined above and R ⁵ is hydrogen, (C ₁ -C ₆ ) alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C ₁ -C ₆ ) alkyl) Independently selected or R ⁶ and R ⁷ together complete an alicyclic or heterocyclic system;

Each D ¹ -D ⁿ is (CR ⁶ R ⁷ ) _Z (wherein R ⁶ and R ⁷ are as defined above);

Each y and z is 0 or an integer from 1 to 10, and the sum y + z is greater than 2 and less than or equal to 10;

Each G ¹ -G ⁿ is -NR ₃ CO-, -NR ³ CS-, -NR ³ SO-, or -NR ³ SO ₂ -in which R ³ is as defined above;

Each A ¹ -A ⁿ and B ¹ -B ⁿ is:

(a ') A is a group of formula (IIa), (IIb) or (IIc), B is N or R ³ N ⁺ , provided that at least one A is a group of formula (IIc); or

(b ') A is a group of formula (IId) and B is CH; or

(c ') A is a group of formula (IIa) or (IIb), B is N or R ³ N ⁺ , provided that at least one y or z is not 1 or 2 and:

(Meal:

X is O, S, Se, NR ³ , CH ₂ or C (CH ₃ ) ₂ ;

Y is a single bond, O, S or NR ₄ ;

Each p and q is 0 or an integer from 1 to 5, and the sum p + q is 10 or less;

Each r and s is 0 or an integer from 1 to 5, and the sum r + s is 10 or less;

Each R ¹ and R ² is independent from the group consisting of hydrogen, hydroxy- or alkoxy- or alkylthio-substituted (C ₁ -C ₄ ) alkyl, hydroxy, alkoxy, alkylthio, amino and halogen Is selected;

Each of R ³ and R ⁴ consists of hydrogen, (C ₁ -C ₄ ) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C ₁ -C ₄ ) alkyl, hydroxy, alkoxy, alkylthio and amino Independently selected from the group consisting of;

Q is -CO ₂ H, -CONR'R ", -SO ₃ H or -SO ₂ NR'R", or an activated derivative of -CO ₂ H or -SO ₃ H;

I is -NHR '"R" "or -NR'" C (O) R "" (where R ', R ", R'" and R "" is hydrogen, alkyl, amino protecting group, reporter ligand, intercalator Is independently selected from the group consisting of chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides and soluble and insoluble polymers.

(a) a polymer of the formula SS-LL-ZZ, wherein SS is the polymeric beads of the invention as described herein, LL is a linker, and ZZ is a chemical group capable of forming a fixed linkage with an amino acid Providing a substrate;

(b) coupling said polymeric substrate with the first amino acid of formula XX via said fixed linkage:

(XX)

[Meal:

L is selected from the group consisting of naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, and optionally the amino group is amino Protected by a protector;

Each C is (CR ⁶ R ⁷ ) _y , wherein R ⁶ is hydrogen and R ⁷ is selected from the group consisting of side chains of naturally occurring alpha amino acids, or R ⁶ and R ⁷ are hydrogen, (C ₂ -C ₆ ) alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C ₁ -C ₆ ) alkoxy, (C ₁ -C ₆ ) alkylthio, NR ³ R ⁴ and SR ⁵ , wherein R ³ and R ⁴ are As defined and R ⁵ is independently selected from the group consisting of hydrogen, (C ₁ -C ₆ ) alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C ₁ -C ₆ ) alkyl) or Or R ⁶ and R ⁷ together form an alicyclic or heterocyclic system;

Each D is (CR ⁶ R ⁷ ) _Z , wherein R ⁶ and R ⁷ are as defined above;

Each y and z is 0 or an integer from 1 to 10, and the sum y + z is greater than 2 and less than or equal to 10;

Each of A and B is:

(a ') A is a group of formula (IIc) and B is N or R ³ N ⁺ ; or

(b ') A is a group of formula (IId) and B is CH; or

(c ') A is a group of formula (IIa) or (IIb), B is N or R ³ N ⁺ , provided that at least one y or z is not 1 or 2 and:

(Meal:

X is O, S, Se, NR ³ , CH ₂ or C (CH ₃ ) ₂ ;

Y is a single bond, O, S or NR ₄ ;

Each p and q is 0 or an integer from 1 to 5, and the sum p + q is 10 or less;

Each r and s is 0 or an integer from 1 to 5, and the sum r + s is 10 or less;

Each R ¹ and R ² is independent from the group consisting of hydrogen, hydroxy- or alkoxy- or alkylthio-substituted (C ₁ -C ₄ ) alkyl, hydroxy, alkoxy, alkylthio, amino and halogen Is selected;

Each of R ³ and R ⁴ consists of hydrogen, (C ₁ -C ₄ ) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C ₁ -C ₄ ) alkyl, hydroxy, alkoxy, alkylthio and amino Independently selected from the group consisting of;

Each E is COOH, CSOH, SOOH, SO ₂ OH or an activated or protected derivative thereof;

Each F is NHR ₃ or NPgR, wherein R ₃ is as defined above and Pg is an amino protecting group;

(c) removing said amino protecting group from the first amino acid coupled to produce a free amino group; And

(d) reacting the free amino group with a second amino acid of Formula (XX) to form a peptide chain. In some embodiments:

(e) removing the amino protecting group from the second amino acid to produce a terminal free amino group on the peptide chain; And

(f) the method further comprises extending the peptide chain by reacting the free amino group on the peptide chain with additional amino acids of formula (XX). In some embodiments, steps e and f are performed several times. Some additional embodiments further comprise removing one or more protecting groups remaining on the amino acid moiety of the peptide chain. Preferably, upon completion of the synthesis of the desired sequence, the anchoring linkage is cleaved from the support without substantially degrading the assembled chain.

In some embodiments of the method, the chemical group capable of forming a fixed linkage is chloro-, bromo- and iodo-substituted alkyl, amino-substituted alkyl, amino and aryl-substituted alkyl, amino- and alkylaryl-substituted Alkyl, hydroxy-substituted alkyl, or derivatives thereof having spacer groups that can be cleaved substantially without cleavage of the polypeptide. In some embodiments, chloro-substituted alkyl is chloromethyl, amino-substituted alkyl is aminomethyl, amino- and alkyl-substituted aryl is α-aminobenzyl, and amino- and alkylaryl-substituted alkyl is α-amino- It is selected from the group consisting of 3- and α-amino-4-methylbenzyl, and the hydroxy-substituted alkyl is hydroxymethyl. In some additional embodiments, the chemical group is derived from an amino-containing moiety selected from amino-substituted alkyl, amino- and aryl substituted alkyl, and amino- and alkylaryl-substituted alkyl; The chemical groups include 4- (haloalkyl) aryl-lower alkanoic acid, Boc-aminoacyl-4- (oxymethyl) aryl-lower alkanoic acid, N-Boc-p-acylbenzhydrylamine, N-Boc-4'- From (lower alkyl) -p-acylbenzhydrylamine, N-Boc-4 '-(lower alkoxy) -p-acylbenzhydrylamine, and 4-hydroxymethylphenoxy-lower alkanoic acid. Derived spacer groups.

In some embodiments, compound X has the formula:

[Meal:

Each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;

Each R ^{7 '} is independently selected from the group consisting of hydrogen and side chains of naturally occurring alpha amino acids;

n is an integer from 1 to 60;

Each k, l, and m is independently 0 or an integer from 1 to 5;

Each p is 0 or 1;

R ^h is OH, NH ₂ or —NHLysNH ₂ ;

R ⁱ is H or COCH ₃ ].

In further embodiments, Compound X has the formula:

[Meal:

Each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;

Each R ^{7 '} is independently selected from the group consisting of hydrogen and side chains of naturally occurring alpha amino acids;

n is an integer from 1 to 60;

Each k, l, and m is independently 0 or an integer from 1 to 5;

Each p is 0 or 1;

R ^h is OH, NH ₂ or —NHLysNH ₂ ;

R ⁱ is H or COCH ₃ ].

In a further embodiment, the amino acid of formula (XX) has the formula

[Meal:

Each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;

Each R ^{7 '} is independently selected from the group consisting of hydrogen and side chains of naturally occurring alpha amino acids;

Each k, l, and m is independently 0 or an integer from 1 to 5;

In a further embodiment, the amino acid of formula (XX) has the formula

[Meal:

Each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;

Each R ^{7 '} is independently selected from the group consisting of hydrogen and side chains of naturally occurring alpha amino acids;

Each k, l, and m is independently 0 or an integer from 1 to 5;

In one embodiment, the present invention is crosslinked, having excellent properties such as bead size distribution, pore size, density, excellent uniformity in swelling properties and / or resistance to solvents and reagents typically used in oligomer synthesis. Provide functionalized polystyrene beads. In some preferred embodiments, the beads are very good in loading properties. In some preferred embodiments, the loading dose of the beads is at least about 50 μmol per gram of beads; At least about 100 μmol per gram of beads; At least about 150 μmol per gram of beads; At least about 200 μmol per gram of beads; At least about 250 μmol per gram of beads; At least about 300 μmol per gram of beads; At least about 350 μmol per gram of beads; At least about 400 μmol per gram of beads; Or at least about 450 μmol per gram of beads. In some embodiments, the loading dose of the beads is about 100 μmol per gram of beads to about 350 μmol per gram of beads.

In some embodiments, the average particle size of the beads of the invention ranges from about 1 μm to about 1000 μm. In a preferred embodiment, the average particle size of the beads of the invention ranges from about 5 μm to about 500 μm. In a particularly preferred embodiment, the average particle size of the beads of the invention ranges from about 10 μm to about 300 μm.

The polymeric bead supports of the present invention can be applied to the preparation of any of a wide range of monomeric and polymeric molecules synthesized by a combination method. Combinatorial libraries are useful per se and such libraries and compounds comprising them are now widely recognized as being of great commercial importance. Indeed, a department of chemistry has been developed to study many commercial aspects of combinatorial libraries. In order to maximize the benefits of each traditional combinatorial approach, new strategies for combinatorial deconvolution have been developed independently by different groups. Selection techniques include peptides (Geysen et al., J. Immun. Meth., 1987, 102, 259; Houghten et al., Nature, 1991, 354, 84; Owens et al., Biochem. Biophys. Res. Commun., 1991, 181, 402; Doyle, PCT WO 94/28424; Brennan, PCT WO 94/27719; Nucleic acids (Wyatt et al., Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 1356; Ecker et al., Nucleic Acids Res., 1993, 21, 1853); Non-peptide and small molecules (Simon et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 9367); Zuckermann et al., J. Am. Chem. Soc, 1992, 114, 10646; Bartlett et al., WO 91/19735; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 10922; DeWitt et al., Proc. Natl. Acad. Sci. USA , 1993, 90, 6909; Cody et al., US Pat. No. 5,324,483; Houghten et al., PCT WO 94/26775; Ellman, US Pat. No. 5,288,514; Still et al., WO 94/08051 Kauffman et al., PCT WO 94/24314; Carell et al., Angew. Chem. Int. Ed. Engl, 1994, 33, 2059; Carell et al., Angew. Chem. Int. Ed. Engel, 1994, 33, 2061; Lebl et al., WO 94/28028]. Each of these documents is incorporated herein by reference in its entirety. A review of the references above indicates that the highest of these techniques is for the selection of peptides and nucleic acids.

Most of the techniques reported to date involve repeated synthesis and screening of even more simplified subsets of oligomers such as peptides and oligonucleotides. Monomers or sub-monomers used include amino acids, amino acid-like molecules, ie carbamate precursors, and nucleotides, both of which are bifunctional. Using this technique, libraries were analyzed for activity in cell-based assays or for binding and / or inhibition of purified protein targets.

In some combinatorial approaches, multifunctional scaffolds with multiple diversity sites are used, and derivatization of these sites into various building blocks to form libraries of various small molecular compounds. Libraries can be generated so that each individual compound can be synthesized and isolated separately, or synthesized and used as a mixture of several desired compounds. Mixtures of compounds can be obtained using mixtures of scaffolds and / or building blocks.

The diversity of combinatorial libraries can vary inherent in the physical and chemical properties of each scaffold and building block used, the number of different building blocks used in each derivatization step, the physical and chemical properties of the bonds arising from the derivatization chemistry, and It is represented by the interaction of folds with building block chemistry. Used together, these interactions provide a unique shape for each individual compound in the combinatorial library.

The polymeric bead supports of the present invention are applicable to the preparation of any of a wide range of monomeric and oligomeric monomers synthesized by such combination methods. These include conventional small molecule drugs, and oligomeric species derived from larger species such as oligomeric peptidomimetic, peptoids, and nucleotides, as well as other preorganized or rigid scaffolds. Molecules are included.

For example, acids, amines and amino acids are a class of building blocks for which their use in combinatorial chemistry is of great importance because of their reactivity with various functional groups and the availability of many such compounds of various structures from commercial sources. For example, amino acids have been widely used in the synthesis of small molecule combinatorial libraries. The use of amino acids as major building blocks in the construction of substituted heterocyclic libraries has been carried out in cyclic elements and in probing libraries for the study of known pharmacophores by various populations ( Bunin and Ellman, J. Am. Chem. Soc, 1992, 114, 10997; DeWitt et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6909; Nefzi, et al., Tetrahedron Lett., 1997, 38, 931; Bartlett, et al., Book of Abstracts, 213th American Chemical Society National Meeting, San Francisco, 1997, American Chemical Society, Washington DC, ORGN-273.

In some combination procedures, the scaffold has a plurality of shielded (ie protected) functional groups (diversity sites). All diversity sites are protected or shielded in such a way that the protection and deprotection scheme is naturally orthogonal, that is, selectively deprotected without affecting the integrity of any other shield. This allows the individual diversity sites to be selectively functionalized when the scaffold is attached or built up during the synthesis of oligomeric or monomeric compounds. Alternatively, it also allows multiple diversity sites to react simultaneously if desired.

Diversity sites can be combined with various building blocks. Sites available for combination include reactive amino and hydroxy groups. Derivatization of scaffolds at the diversity site includes various but not limited to carboxylic acids, acid halides, anhydrides, sulfonic acids, sulfonyl halides, isocyanates, isothiocyanates, ketones, aldehydes, amines and amino acids. This is accomplished using blocks.

In some embodiments, the present invention provides for the addition of functional groups on a monocyclic or bicyclic scaffold attached to the solid support of the present invention. The preparation of combinatorial libraries begins with a monocyclic or bicyclic scaffold attached to a solid support either directly or through a linker that is stable to synthetic conditions but cleaved at the end of the synthesis to release the compound into solution. Preferred linkers include esters, especially those derived from succinic acid. Alternatively, the scaffold can be coupled to a constant moiety attached to the support, such as DMT, ethylene glycol or similar diols.

Scaffolds can be uniform or can be structurally diverse populations of monocyclic and / or bicyclic ring systems that provide several relative orientations of functional groups and various substituents suspended. Additional combination sites may be present on the scaffold in the form of protected hydroxy or amino groups or other masked functional groups that can optionally react with the building block if desired. Scaffolds and building blocks used in combinatorial libraries have several functional groups that are used together to provide various properties (“diversity”) to the resulting library members. Such functional groups include hydrogen-bond donors and acceptors, ionic moieties, polar moieties, hydrophobic moieties, aromatic moieties, and electron-donors and acceptors. Together, the properties of the individual scaffolds and building blocks contribute to the uniqueness of the individual compounds in which they are found. Thus, a library of such compounds will have a myriad of properties, ie "diversity". Collectively, the nature of the individual scaffolds and building blocks that together form the individual library compound contributes to the uniqueness of the compound and confers it specific properties for interaction with cellular, enzymatic or nucleic acid target sites.

As used herein, the term oligonucleotide means an oligomer having m subunits included in parentheses [] of the formula:

Oligonucleotide

[Other variables in the formula are defined above and described in more detail below]. While the oligonucleotides produced are shown in single stranded form, it is understood that oligonucleotides are used in double stranded form. For example, in an antisense method commonly referred to as siRNA, two strands of RNA or RNA-like oligonucleotides are prepared and annealed together, with two nucleotides often overlapping at the ends. Thus, the construction of both single stranded and double stranded oligonucleotides is implemented in the present invention.

Nuclear base

Nucleobases Bx may be the same or different and include naturally occurring nucleobases adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C), as well as modified nucleobases . Modified nucleobases include heterocyclic moieties that are structurally related to a naturally-occurring nucleobase but are chemically modified to impart some properties that the naturally-occurring nucleobase does not have. The term “nucleobase” as used herein is intended to be synonymous with “nucleic acid base or mimetic thereof”. In general, a nucleobase is any structure that contains one or more atoms or groups of atoms capable of hydrogen bonding to the base of an oligonucleotide.

As used herein, the term "unmodified" or "natural" nucleobase includes purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U) Included. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-mC), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine and other Alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH ₃ ) uracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol , 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-aguauanine and 8-azadenine, 7-deazaguanine and 7-deazaadenin It includes 3-de-aza and 3-guanine to adenine aza. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido [5,4-b] [1,4] benzoxazine-2 (3H) -one), phenothiazine cytidine ( 1H-pyrimido [5,4-b] [1,4] benzothiazine-2 (3H) -one), G-clamps such as substituted phenoxazine cytidines (eg 9- (2- Aminoethoxy) -H-pyrimido [5,4-b] [1,4] benzoxazine-2 (3H) -one), carbazole cytidine (2H-pyrimido [4,5-b] indole 2-one), pyridoindole cytidine (H-pyrido [3 ', 2': 4,5] pyrrolo [2,3-d] pyrimidin-2-one). Modified nucleobases include those in which purine or pyrimidine bases have been replaced by other heterocycles such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Additional nucleobases include those disclosed in US Pat. No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289- 302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993.

Some such nucleobases are particularly useful for increasing the binding affinity of oligomeric compounds of the invention. This includes 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine do. 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2 ° C. (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), which are presently preferred base substitutions, and more particularly preferred when combined with 2'-0-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include U.S. Pat. 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; And 5,681,941, some of which are co-owned with the present application and incorporated herein by reference, respectively, and US Patent 5,750,692, which is co-owned with and also incorporated herein by reference. Does not.

Further modifications may also be made at other positions on the oligonucleotide, in particular at the 3 'position of the sugar on the 3' terminal nucleotide and the 5 'position of the 5' terminal nucleotide. For example, one further modification of an oligonucleotide of the invention wherein the ligand is conjugated involves chemically modifying one or more additional non-ligand moieties or conjugates that enhance the oligonucleotide's activity, cellular distribution, or cellular uptake. It is accompanied by the connection. Such moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), bile acids (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306); Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3, 2765]), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), aliphatic chains, for example Dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111); Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49]), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phospho Nate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), polyamines or polyethylene glycols Cole chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), palmityl moieties ( Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923), but is not limited to such.

Representative US patents taught to prepare such oligonucleotide conjugates include US Pat. No. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, including, but not limited to, some of which are co-owned with the present application and incorporated herein by reference, respectively.

In some embodiments of the invention, oligomeric compounds, such as oligonucleotides, are prepared having polycyclic heterocyclic compounds instead of one or more heterocyclic base moieties. Many tricyclic heterocyclic compounds have been reported in the past. Such compounds are routinely used in antisense applications, increasing the binding properties of the modified strand to the target strand. The most studied variant targets guanosine, so it has been termed a G-clamp or cytidine analog. Many such polycyclic heterocyclic compounds have the formula:

Representative cytosine analogs that form three hydrogen bonds with guanosine in the second strand include 1,3-diazphenoxazin-2-one (R ₁₀ = O, R ₁₁ -R ₁₄ = H) (Kurchavov, et al. , Nucleosides and Nucleotides, 1997, 16, 1837-1846]), 1,3-diazapenothiazin-2-one (R ₁₀ = S, R ₁₁ -R ₁₄ = H) ([Lin, K.-Y. Jones, RJ; Matteucci, MJ Am. Chem. Soc. 1995, 117, 3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazin-2-one (R ₁₀ = O, R ₁₁ -R ₁₄ = F) (Wang, J .; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388). Such base modifications incorporated into oligonucleotides have also been shown to hybridize with complementary guanine, and the latter also hybridize with adenine to enhance helical thermal stability by expanded stacking interactions (with the present application Co-owned and incorporated herein by reference in its entirety, entitled “Modified Peptide Nucleic Acids” and US Patent Application Serial No. 10 / 155,920, filed May 24, 2002; and titled “Nuclease Resistant Chimeric Oligonucleotides, "US Patent Application Serial No. 10 / 013,295, filed May 24, 2002).

Cytosine analogs / substituents are bound to the rigid 1,3-diazaphenoxazine-2-one scaffold (R ₁₀ = O, R ₁₁ = -O- (CH ₂ ) ₂ -NH ₂ , R _{12 -14} = H) Additional helix-stabilizing properties were observed when having an attached aminoethoxy moiety (Lin, K.-Y .; Matteucci, MJ Am. Chem. Soc. 1998, 120, 8531-8532). Binding studies have shown that single incorporation can enhance the binding affinity of a model oligonucleotide to its complementary target DNA or RNA to ΔT _m up to 18 ° relative to 5-methyl cytosine (dC5 ^me ). It is the best known affinity enhancer for modification. On the other hand, the gain in helical stability does not impair the specificity of the oligonucleotide. T _m data show a greater difference between complete match and mismatched sequences compared to dC5 ^me . It has been proposed that the tethered amino group acts as an additional hydrogen bond donor that interacts with the Hoogsteen face of the complementary guanine, ie O6, to form four hydrogen bonds. This means that the increased affinity of the G-clamp is mediated by the combination of expanded base stacking and additional specific hydrogen bonds.

Additional tricyclic heterocyclic compounds applicable to the present invention and methods of using the same are disclosed in US Pat. No. 6,028,183, issued May 22, 2000, and US Pat. No. 6,007,992, issued December 28, 1999. The contents of which are generally assigned to this application and incorporated herein by reference in their entirety. Such compounds include those having the formula:

Wherein R ₁₁ includes (CH ₃ ) ₂ N— (CH ₂ ) ₂ —O—; H ₂ N- (CH ₂ ) _3- ; Ph-CH ₂ -OC (= 0) -N (H)-(CH ₂ ) _3- ; H2N-; Fluorophenyl-CH ₂ -OC (= 0) -N (H)-(CH ₂ ) _3- ; Phthalimidyl-CH ₂ -OC (= 0) -N (H)-(CH ₂ ) _3- ; Ph—CH ₂ —OC (═O) —N (H) — (CH ₂ ) ₂ —O—; Ph—CH ₂ —OC (═O) —N (H) — (CH ₂ ) ₃ —O—; (CH ₃ ) ₂ N—N (H) — (CH ₂ ) ₂ —O—; Fluorenyl-CH ₂ -OC (= 0) -N (H)-(CH ₂ ) ₂ -O-; Fluorenyl-CH ₂ -OC (= 0) -N (H)-(CH ₂ ) ₃ -O-; H ₂ N- (CH ₂ ) ₂ -O-CH _2- ; N _3- (CH ₂ ) ₂ -O-CH _2- ; H ₂ N— (CH ₂ ) ₂ —O—, and NH ₂ C (═NH) NH—.

Also disclosed are tricyclic heterocyclic compounds of the formula:

[Meal:

R _10a is O, S or N—CH ₃ ; R _11a is A (Z) _x1 , wherein A is a spacer and Z is a label bonding group optionally bonded to an independently detectable label, but R _11a is not an amine, protected amine, nitro or cyano; X 1 is 1, 2 or 3; R _b is independently -CH =, -N =, -C ( C 1 -8 alkyl) = or -C (halogen) =, but the adjacent R _b or not both -N =, or two adjacent R _b Together to form a ring of the formula:

Wherein R _c is not independent as -CH =, -N =, -C ( C 1 -8 alkyl) = or -C (halogen) =, but the adjacent Rb both -N =].

The enhanced binding affinity of phenoxazine derivatives, together with intact sequence specificity, makes them useful nucleobase analogs for the development of stronger antisense-based drugs. Indeed, promising data have been derived from in vitro experiments indicating that heptanucleotides containing phenoxazine substitutions can activate RNAase H, enhance cellular uptake, and exhibit increased antisense activity (Lin, K.-Y .; Matteucci, MJ Am. Chem. Soc. 1998, 120, 8531-8532]. As single substitutions have been shown to significantly improve the in vivo efficacy of 20-mer 2'-deoxyphosphothioate oligonucleotides, activity enhancement is even more pronounced for G-clamps (Flanagan, WM; Wolf, JJ Olson, P .; Grant, D .; Lin, K.-Y .; Wagner, RW; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, in order to optimize the oligonucleotide design and to better understand the impact of such heterocyclic modifications on biological activity, it is important to evaluate its effect on the nuclease stability of the oligomer.

Additional tricyclic and tetracyclic heteroaryl compounds applicable to the present invention include those having the formula:

Wherein R ₁₄ is NO ₂ or both R ₁₄ and R ₁₂ are independently —CH ₃ . The synthesis of such compounds is described in US Pat. No. 5,434,257 issued July 18, 1995, US Pat. No. 5,502,177 issued March 26, 1996, and US Pat. No. 5,646, 269 (8 July 1997). The contents of which are generally assigned to this application and incorporated herein in their entirety.

Additional tricyclic heterocyclic compounds applicable to the present invention are also disclosed in the "257, 177 and 269" patents and include those having the formula: tautomers, solvates or salts thereof:

[Where a and b are independently 0 or 1 and the sum of a and b is 0 or 1; A is N, C or CH; X is S, O, C═O, NH or NCH ₂ , R ⁶ ; Y is C═O; Z together with A forms an aryl or heteroaryl ring structure comprising 5 or 6 ring atoms, wherein the heteroaryl ring is 1 O ring heteroatom, 1 N ring heteroatom, 1 S ring heteroatom, carbon One O and one N ring heteroatom separated by atoms, one S and one N ring heteroatom separated by C atoms, two N ring heteroatoms separated by carbon atoms, or two or more One or more ring carbon atoms containing three N ring heteroatoms separated by this carbon atom, wherein the aryl or heteroaryl ring carbon atom is unsubstituted or bridged to something other than H is R ²⁰ or = O Substituted; Or Z together with A forms an aryl ring structure comprising six ring atoms, wherein the aryl ring carbon atom is one or more ring carbon atoms which are not substituted or bridged with other than H to R ⁶ or ═O. Substituted; R ⁶ is independently H, C _{1 -6} alkyl, C _{2 -6} alkenyl, C _{2 -6-alkynyl, NO 2, N (R 3} ) 2, or CN, or halo, or R ⁶ is adjacent to the group Z Completing the phenyl ring with R ⁶ ; R ²⁰ is independently H, C _{1 -6} alkyl, C _{2 -6} alkyl, C _{2 -6} alkenyl, C _{2 ^{-6-alkynyl, NO 2, N (R 21}} ) 2, CN, or is halo, or R ²⁰ together with adjacent R ²⁰ completes the ring containing 5 or 6 ring atoms; R ²¹ is independently H or a protecting group; R ³ is a protecting group or H].

More specific examples of bases included in the "257, 177 and 269" patents are compounds of the formula:

Wherein each R ₁₆ is independently selected from hydrogen and various substituent groups.

Additional polycyclic base moieties having the following formula are disclosed in US Patent Application Serial No. 09 / 996,292 (filed November 28, 2001), co-owned with and incorporated herein by reference:

[Wherein A ₆ is O or S; A ₇ is CH ₂ , N—CH ₃ , O or S; Each of A ₈ and A ₉ are hydrogen, or one of, or A ₈ and A ₉ is hydrogen and the other one of A ₈ and A ₉ are selected from the group consisting of:

Wherein G is -CN, -OA ₁₀ , -SA ₁₀ , -N (H) A ₁₀ , -ON (H) A ₁₀ or -C (= NH) N (H) A ₁₀ ; Q ₁ is H, -NHA ₁₀ , -C (O) N (H) A ₁₀ , -C (= S) N (H) A ₁₀ or -C (= NH) N (H) A ₁₀ ; each Q ₂ is independently H or Pg; A ₁₀ is H, Pg, substituted or unsubstituted C ₁ -C ₁₀ alkyl, acetyl, benzyl,-(CH ₂ ) _p3 NH ₂ ,-(CH ₂ ) _p3 N (H) Pg, D or L α-amino acids or peptides derived from D, L or racemic α-amino acids; Pg is a nitrogen, oxygen or thiol protecting group; each p1 is independently 2 to about 6; p2 is 1 to about 3 p3 is 1 to about 4).

Sugars and Sugar Substitutions

Sugar moieties of the formula: represent sugar portions of common nucleosides or nucleotides as embodied by the present invention:

[Each dotted line (----) in the formula indicates an attachment point to an adjacent phosphorus atom.

Suitable 2'-substituents corresponding to R ' ₂ include OH, F, O-alkyl (eg O-methyl), S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl ; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl- O-alkyl is included, wherein alkyl, alkenyl and alkynyl are substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl or alkynyl, respectively. O [(CH ₂ ) _g O] _h CH ₃ , O (CH ₂ ) _g OCH ₃ , O (CH ₂ ) _g NH ₂ , O (CH ₂ ) _g CH ₃ , O (CH ₂ ) _g ONH ₂ , and Particular preference is given to O (CH ₂ ) _g ON [(CH ₂ ) _g CH ₃ ] ₂ , wherein g and h are from 1 to about 10. Other preferred oligonucleotides include one of the following at the 2 ′ position: C ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocyclo Alkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cutters, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacokinetic properties of oligonucleotides , And other substituents with similar properties. Preferred 2'-modifications include 2'-methoxyethoxy (also known as 2'-0-CH ₂ CH ₂ OCH ₃ , 2'-0- (2-methoxyethyl) or 2'-MOE). (Martin et al., Helv. Chim, Acta, 1995, 78, 486-504). Further preferred variations include 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, ie O (CH ₂ ) ₂ ON (CH ₃ ) ₂ groups, and the following 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), also described in the examples, ie 2'-0-CH ₂ -O-CH ₂ -N (CH ₃ ) ₂ is included.

Other preferred variations include 2'-methoxy (2'-O-CH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ), 2'-allyl (2'-CH _2- CH = CH ₂ ), 2'-0-allyl (2'-0-CH ₂ -CH = CH ₂ ) and 2'-fluoro (2'-F). The 2'-modification can be in the arabino (up) or ribo (down) position. Preferred 2'-arabino modification is 2'-F. Similar modifications can also be made at another position on the oligonucleotide, in particular at the 3 'position of the sugar on the 3' terminal nucleotide or at the 2'-5 'linked oligonucleotide and 5' position of the 5 'terminal nucleotide.

Additional exemplary substituent groups include groups of formula la or lla:

[Wherein R _b is O, S or NH; R _d is a single bond, O or C (═O); R _e is C ₁ -C ₁₀ alkyl, N (R _k ) (R _m ), N (R _k ) (R _n ), N = C (R _p ) (R _q ), N = C (R _p ) ( R _r ) or of formula IIIa:

Has].

Each R _s , R _t , R _u and R _v is independently hydrogen, C (O) R _W , substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or Unsubstituted C ₂ -C ₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, chemical functional or conjugated groups (substituent groups are hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl , Aryl, alkenyl and alkynyl); Or optionally, R _u and R _v together with the nitrogen atom to which they are attached form a phthalimido moiety; Each R _w is independently substituted or unsubstituted C ₁ -C ₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2 -(Trimethylsilyl) -ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl; R _k is hydrogen, a nitrogen protecting group or -R _x -R _y ; R _p is hydrogen, a nitrogen protecting group or -R _x -R _y ; R _x is a bond or linking moiety; R _y is a chemical functional group, conjugated group or a solid support medium; Each R _m and R _n is independently H, a nitrogen protecting group, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl ( wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, ^{_{alkynyl), NH 3 +, N (}} R u) (R _v ), guanidino and acyl, wherein acyl is an acid amide or ester; Or R _m and R _n together are a nitrogen protecting group or are linked in a ring structure optionally comprising an additional heteroatom selected from N and O, or a chemical functional group; R _j is OR _z , SR _z , or N (R _z ) ₂ ; Each R ₂ is independently H, C ₁ -C ₈ alkyl, C ₁ -C ₈ haloalkyl, C (═NH) N (H) R _u , C (═O) N (H) R _u or OC ( ═O) N (H) R _u ; R _f , R _g and R _h include ring systems having from about 4 to about 7 carbon atoms or from about 3 to about 6 carbon atoms and one or two heteroatoms selected from oxygen, nitrogen and sulfur, wherein The ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic.

R _j is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, having 6 to about 14 carbon atoms Aryl, N (R _k ) (R _m ) OR _k , halo, SR _k or CN; m _a is from 1 to about 10; Each mb is independently 0 or 1; mc is 0 or an integer from 1 to 10; md is an integer from 1 to 10; me is 0, 1 or 2; However, when mc is 0, md exceeds 1.

Representative substituent groups of Formula I are disclosed in US Pat. No. 6,172,209. Representative cyclic substituent groups of formula (II) are disclosed in US Pat. No. 6,271,358.

Particularly useful sugar substituent groups include O [(CH ₂ ) _g O] _h CH ₃ , O (CH ₂ ) _g OCH ₃ , O (CH ₂ ) _g NH ₂ , O (CH ₂ ) _g CH ₃ , 0 (CH ₂ ) _g ONH ₂ and O (CH ₂ ) _g ON [(CH ₂ ) _g CH ₃ )] ₂ (where g and h are from 1 to about 10).

Some particularly useful oligomeric compounds of the invention contain one or more nucleosides having one of the following substituent groups: C ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O -Alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , hetero Cycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cutters, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or pharmacokinetic properties of oligonucleotides Groups to improve, and other substituents with similar properties. Preferred variations include 2'-methoxyethoxy [also known as 2'-0-CH ₂ CH ₂ OCH ₃ , 2'-0- (2-methoxyethyl) or 2'-MOE] (Martin et al. , Helv. Chim. Acta, 1995, 78, 486], ie, alkoxyalkoxy groups. A further preferred variant is the O (CH ₂ ) ₂ ON (CH ₃ ) ₂ group, also known as 2'-dimethylaminooxyethoxy, ie 2'-DMAOE. Exemplary aminooxy substituent groups are described in US Pat. Appl. Serial No. 09 / 344,260, filed Jun. 25, 1999, entitled “Aminooxy-Functionalized Oligomers”, which is incorporated herein by reference in its entirety; And US patent application Ser. No. 09 / 370,541, filed August 9, 1999 and entitled “Aminooxy-Functionalized Oligomers and Methods for Making Same”.

Other particularly advantageous 2'-modifications include 2'-methoxy (2'-O-CH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ) and 2'-fluoro (2 '-F) is included. Similar modifications result in another position on the nucleoside and oligomer, in particular the 3 'of a nucleoside such as a 2'-5' linkage oligomer having a bond from the 3 'position or from the 2'-position of a sugar on the 3' terminal nucleoside. And at the 5 'position of the 5' terminal nucleotide. The oligomers may also have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative US patents taught to produce such modified sugar structures include US Pat. No. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; And 5,700,920, some of which are co-owned and each incorporated herein by reference, and co-owned US patent application 08 / 468,037, filed Jun. 5, 1995, and also incorporated herein by reference. However, it is not limited to this.

Representative guanidino substituent groups shown in Formulas (III) and (IV) are co-owned US Patent Application 09 /, entitled “Functionalized Oligomers”, filed July 7, 1999, and paying a registration fee on October 23, 2002. 349,040.

Representative acetamido substituent groups are disclosed in US Pat. No. 6,147,200, which is incorporated herein by reference in its entirety. Representative dimethylaminoethyloxyethyl substituent groups are disclosed in the international patent application PCT / US99 / 17895, filed August 6, 1999, entitled “2'-O-Dimethylaminoethyloxyethyl-Modified Oligonucleotides," which is incorporated by reference in its entirety. Included herein. In such nucleosides comprising pentofuranosyl sugars, phosphate groups can be linked to the 2 ', 3' or 5 'hydroxyl moieties of the sugar. In the formation of oligonucleotides, phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound. While each end of this linear polymeric structure can be connected by hybridization or by the formation of a covalent bond, an open linear structure is generally preferred. Within the oligonucleotide structure, phosphate groups are generally referred to as forming internucleoside bonds of oligonucleotides. Normal nucleoside linkages between RNA and DNA are phosphodiester bonds from 3 'to 5'.

Although the present invention can be applied to produce oligonucleotides for any desired end use (eg as probes for use in polymerase chain reaction), one preferred use of oligonucleotides is in antisense therapy. One mode of action often used in antisense therapies is the so-called RNAase H mechanism in which DNA strands are introduced into cells, which hybridize to the RNA strands. DNA-RNA hybrids are recognized by restriction enzyme RNAase H, which cuts the RNA strand. In normal cases, the RNA strand is messenger RNA (mRNA), which, after cleavage, cannot be translated into the corresponding peptide or protein sequence in ribosomes. In this way, DNA can be used as an agent to regulate the expression of a particular gene.

It has been found that by incorporating short stretches of DNA into oligonucleotides, RNAase H mechanisms can be effectively used to modulate expression of target peptides or proteins. In some embodiments of the invention, oligonucleotides incorporating DNA stretch and stretch of RNA or 2'-modified RNA can be used to effectively regulate gene expression. In a preferred embodiment, the oligonucleotide comprises a DNA construct flanked by two 2'-modified RNA stretches. Preferred 2'-modifications include 2'-MOE as described herein.

Ribosyl sugar moieties were also extensively studied to assess the effect of modification on the properties of oligonucleotides as compared to unmodified oligonucleotides. The 2'-position of the sugar moiety is one of the most studied sites for modification. Certain 2'-substituent groups have been shown to increase lipophilic properties and enhance properties such as binding affinity, chemical stability and nuclease resistance of oligonucleotides to target RNA. Many modifications at the 2′-position, which show enhanced binding affinity, also allow the sugar rings to be C ₃ -endo shaped.

RNA exists as what is termed the "A-type" geometry, and DNA exists as the "B-type" geometry. In general, RNA: RNA duplexes are more stable than DNA: DNA duplexes or have a higher melting point (Tm) (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, NY.); Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634. Increased stability of RNA has resulted in several structural features, and most notably in base stacking interactions generated from A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21 , 2051-2056]. The presence of 2 'hydroxyls in the RNA causes the sugar to be deflected into a C3' enducker, ie a fuker also named Northern Fuker, which causes the duplex to prefer A-type geometry. Deoxy nucleic acids, on the other hand, prefer C2 'endo sugar fukers, ie, fukers, also known as Southern fukers, which are believed to confer less stable B-form geometry. (Sanger, W. ( 1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, NY]. In addition, the 2 'hydroxyl group of RNA can form a network of hydrogen bonds mediated by water, which helps to stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 55, 8489-8494). ).

However, DNA: RNA hybrid duplexes are generally less stable than pure RNA: RNA duplexes, and may be more or less stable than DNA: DNA duplexes, depending on their sequence (Searle et al., Nucleic Acids Res., 1993). , 21, 2051-2056]. The structure of the hybrid duplex is halfway between the A- and B-shape geometries, which can lead to poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306). Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533]. The stability of DNA: RNA hybrids is central to antisense therapies because the mechanism requires that modified DNA strands bind to mRNA strands. In order to effectively inhibit mRNA, antisense DNA must have a very high binding affinity with mRNA. Otherwise, the desired interaction between the DNA and the target mRNA strands rarely occurs, thereby reducing the efficacy of the antisense oligonucleotides.

Various synthetic modifications have been proposed to improve nuclease resistance or to enhance the affinity of an antisense strand for its target mRNA (Crooke et al., Med. Res. Rev., 1996, 16, 319-344 De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374). Various modified phosphorus-containing bonds have been studied as replacements for natural, readily degraded phosphodiester bonds in oligonucleotides. In general, most of these, such as phosphorothioate, phosphoramidate, phosphonate and mospoodithioate, resulted in oligonucleotides with reduced binding to complementary targets and reduced hybrid stability. .

RNA exists as what is termed the "A-type" geometry, and DNA exists as the "B-type" geometry. In general, RNA: RNA duplexes are more stable than DNA: DNA duplexes or have a higher melting point (Tm) (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, NY.); Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634. Increased stability of RNA has resulted in several structural features, and most notably in base stacking interactions generated from A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21 , 2051-2056]. The presence of 2 = hydroxyl in the RNA causes the sugar to be deflected into a C3 = endo fuker, ie a fuker also named Northern fuker, which causes the duplex to prefer A-type geometry. Deoxy nucleic acids, on the other hand, prefer C2 'endo sugar fukers, ie fukers, also known as Southern fukers, which are believed to confer less stable B-type geometry (Sanger, W. (1984) Principles). of Nucleic Acid Structure, Springer-Verlag, New York, NY]. In addition, 2 = hydroxyl groups of RNA can form a network of hydrogen bonds mediated by water, which helps to stabilize RNA duplexes (Egli et al., Biochemistry, 1996, 55, 8489-8494). ).

However, DNA: RNA hybrid duplexes are generally less stable than pure RNA: RNA duplexes, and may be more or less stable than DNA: DNA duplexes, depending on their sequence (Searle et al., Nucleic Acids Res., 1993). , 21, 2051-2056]. The structure of the hybrid duplex is halfway between the A- and B-shape geometries, which can lead to poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306). Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533]. The stability of DNA: RNA hybrids is a major aspect of antisense therapies because the proposed mechanism requires that the modified DNA strands bind to the mRNA strands. Ideally, antisense DNA should have a very high binding affinity with mRNA. Otherwise, the desired interaction between the DNA and the target mRNA strands rarely occurs, thereby reducing the efficacy of the antisense oligonucleotides.

One synthetic 2'-modification that confers increased nuclease resistance and very high binding affinity for nucleotides is 2 = -methoxyethoxy (MOE, 2'-OCH ₂ CH ₂ OCH ₃ ) side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000; Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443). One immediate advantage of MOE substitutions is an improvement in binding affinity, which is greater than many similar 2 'modifications such as O-methyl, 0-propyl, and O-aminopropyl (Freier and Altmann, Nucleic Acids Research, (1997) 25: 4429-4443]. 2 = -O-methoxyethyl-substituted oligonucleotides have also been shown to be antisense inhibitors of gene expression with promising characteristics for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486- 504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926]. Compared with DNA, it shows improved RNA affinity and higher nuclease resistance. Chimeric oligonucleotides with 2═-O-methoxyethyl-ribonucleoside wings and a central DNA-phosphothioate window have also been shown to effectively inhibit tumor growth in animal models at low doses. MOE substituted oligonucleotides have shown promising prospects as antisense agents in several disease states. One such MOE substituted oligonucleotide is currently being studied in clinical trials for the treatment of CMV retinitis.

LNAs (oligonucleotides whose 2 ′ and 4 ′ positions are linked by a bridge) also form a duplex with complementary DNA, RNA or LNA with high thermal stability. Circular dichroism (CD) spectra indicate that duplexes (particularly LNA: RNA) with fully modified LNA are structurally similar to A-form RNA: RNA duplexes. Nuclear magnetic resonance (NMR) irradiation of the LNA: DNA duplex confirmed the 3'-endo shape of the LNA monomer. Recognition of double stranded DNA has also been demonstrated, suggesting strand invasion by LNA. Studies of mismatched sequences show that LNA follows the Watson-Crick nucleotide pairing rule and generally improves selectivity over corresponding unmodified reference strands.

In LNAs where the 2'-hydroxyl group is linked to the 4 'carbon atom of the sugar ring, a 2'-C, 4'-C-oxymethylene bond is formed, thus forming a bicyclic sugar moiety. The bond may be a methylene (—CH ₂ —) _n group bridging the 2 ′ oxygen atom and the 4 ′ carbon atom, where n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs exhibit very high duplex thermal stability (Tm = + 3 to + 10 ° C.) with complementary DNA and RNA, stability to 3′-exonucleolytic degradation and good solubility properties. Other preferred bridge groups include 2'-deoxy-2'-CH ₂ OCH _2-4 'bridges.

Alternative  Linker

In addition to phosphate diester and phosphorothioate diester linkages, further linkers are known in the art. The primary interest of the present invention should be directed to phosphate diesters and phosphorothioate diester oligonucleotides, but are chimeric compounds having more than one type of bond, as well as non- as described in more detail below. Oligomers having phosphate / phosphothioate diester bonds are also embodied, in whole or in part, in the context of the present invention.

Exemplary non-phosphate / phosphothioate diester bonds embodied within the art include: phosphorodithioate, phosphoester, aminoalkylphosphotriester, methyl and other alkyl phosphors Nates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (3'-amino phosphoramidates and aminoalkyls) Phosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates. Additional bonds include the following: thiodiester (-OC (O) -S-), thionocarbamate (-OC (O) (NJ) -S-), siloxane (-O-Si (J ) ₂ -O-), carbamate (-OC (O) -NH- and -NH-C (O) -O-), sulfamate (-OS (O) (O) -N- and -NS ( O) (O) -N-, morpholino sulfamide (-OS (O) (N (morpholino)-), sulfonamide (-O-SO ₂ -NH-), sulfide (-CH _2- S-CH _2- ), sulfonate (-O-SO ₂ -CH _2- ), N, N'-dimethylhydrazine (-CH ₂ -N (CH ₃ ) -N (CH ₃ )-), thioformacetal (-S-CH ₂ -O-), formacetal (-O-CH ₂ -O-), thioketal (-SC (J) ₂ -O-), ketal (-OC (J) ₂ -O- ), Amine (-NH-CH ₂ -CH _2- ), hydroxylamine (-CH ₂ -N (J) -O-), hydroxylimine (-CH = NO-), and hydrazinyl (-CH ₂ -N (H) -N (H)-).

In each of these substructures relating to internucleoside linkages, J typically represents a substituent group that is a more complex group that varies depending on the hydrogen or alkyl group or type of linkage.

In addition to linking groups as described above involving the modification or substitution of the -OPO- atoms of naturally occurring bonds, linkages comprising not only 5'-methylene groups but also modifications of one or more -OPO- atoms are within the scope of the present invention. Included. Linkages of this type are well documented in the art and include, but are not limited to the following: amides (-CH ₂ -CH ₂ -N (H) -C (O)) and -CH ₂ -ON = CH- ; And alkyl (-C (J) ₂ -P (= 0) (OJ) -C (J) ₂ -C (J) _2- ). J is as described above.

Oligonucleotide Synthesis

As described above, on support media, for example solid support media, oligonucleotides are generally prepared. In general, oligonucleotides are synthesized by first attaching syntheses (eg, monomers, such as nucleosides) to the support medium first, followed by successive coupling of monomers to the support-binding syntheses. This repetitive elongation eventually results in the final oligomeric compound or other polymer such as a polypeptide. Suitable support media may be soluble or insoluble, or may vary in solubility in various solvents such that the growing support-binding polymer is within or outside of the solution as desired. Traditional support media such as solid support media are mostly insoluble and are typically placed in a reaction vessel while reagents and solvents react with and / or wash the growth chain until the oligomer reaches the target length, after which the oligomer is cleaved from the support. And further worked up, if necessary, to produce the final polymer compound. More recent approaches have introduced soluble supports, including soluble polymer supports, to allow precipitation and dissolution of repeatedly synthesized products at desired times during synthesis (Gravert et al., Chem. Rev., 1997, 97 489-510).

The term support medium is intended to include all forms of support known in the art for the synthesis of oligomeric compounds and related compounds such as peptides. Some representative support media applicable to the present invention include, but are not limited to, the following: controlled pore glass (CPG); Oxalyl-controlled pore glass (see, eg, Alul, et al., Nucleic Acids Research 1991, 19, 1527); Silica-containing particles such as porous glass beads and silica gel such as trichloro- [3- (4-chloromethyl) phenyl] propylsilane formed by reaction of porous glass beads (Parr and Grohmann, Angew. Chem. Internal Ed. 1972, 11, 314, sold by Waters Associates (Framingham, Mass., USA) under the trade name “PORASIL E”; Mono esters of 1,4-dihydroxymethylbenzene and silica (see Bayer and Jung, Tetrahedron Lett, 1970, 4503, sold by Waters Associates under the trade name "BIOPAK"); TENTAGEL (see, eg, Wright, et al., Tetrahedron Letters 1993, 34, 3373); Crosslinked styrene / divinylbenzene copolymer, beaded matrix or POROS, a copolymer of polystyrene / divinylbenzene (available from Perceptive Biosystems); Soluble support medium, polyethylene glycol PEG (see Bonora et al., Organic Process Research & Development, 2000, 4, 225-231).

Additional support media applicable to the present invention include PEPS supports which are polyethylene (PE) films with pendant long chain polystyrene (PS) grafts (molecular weights of the order of 10 ⁶ (Berg, et al., J. Am. Chem. Soc, 1989, 111, 8024 and international patent application WO 90/02749)). The loading capacity of the film is as high as that of the beaded matrix, and additional flexibility allows multiple synthesis to occur simultaneously. PEPS films can be in the form of separate, labeled sheets, each functioning as a separate compartment. During all the same steps of the synthesis cycle, the sheets are held together in a single reaction vessel, allowing simultaneous preparation of multiple peptides at a rate similar to that of a single peptide by conventional methods. In addition, experiments with PEPS polymers of other geometries such as, for example, nonwovens, knitted fabrics, sticks or microwell plates did not show any limitations in synthetic efficiency.

Additional support media applicable to the present invention include crosslinking with N, N'-bisacryloylethylenediamine comprising known amounts of Nt-butoxycarbonyl-beta-alanyl-N'-acryloylhexamethylenediamine. Particles based on copolymers of dimethylacrylamide are included without limitation. Several spacer molecules are typically added via a beta alanyl group followed by the addition of amino acid residue subunits. In addition, the beta alanyl-containing monomer can be replaced with acryloyl safcosine monomer during the polymerization to form the resin beads. After polymerization, the reaction between the beads and ethylenediamine is followed to form resin particles containing a primary amine as a covalently bonded functional group. Polyacrylamide-based supports are relatively more hydrophilic than polystyrene-based supports and are generally used with polar aprotic solvents, including dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and the like (Atherton, et al. , J. Am. Chem. Soc, 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351, and JCS Perkin I 538 (1981).

Additional support media applicable to the present invention include, but are not limited to, composites of resins and another material that is also substantially inert to the organic synthesis reaction conditions used. One exemplary composite (see Scott, et al., J. Chrom. Sci., 1971, 9, 577) uses glass particles coated with a hydrophobic crosslinked styrene polymer containing reactive chloromethyl groups, which is a Northgate Laboratories, Inc. (Hamden, Conn., USA). Another exemplary composite contains a core of polystyrene grafted fluorinated ethylene polymer (Kent and Merrifield, Israeli Chem. 1978, 17, 243) and van Rietschoten in Peptides 1974, Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp. 113-116). Contact solid support media other than PEPS, such as cotton sheets (Lebl and Eichler, Peptide Res. 1989, 2, 232) and hydroxypropylacrylate-coated polypropylene membranes (Daniels, et al., Tetrahedron Lett 1989, 4345). Polyethylene-rod and 96-microtiter wells grafted with acrylic acid to fix growth peptide chains and perform compartmental synthesis. (Geysen, et al., Proc. Natl. Acad. Sci. USA, 1984, 81, 3998). "Tea bags" containing traditionally used polymer beads. (Houghten, Proc. Natl. Acad. Sci. USA, 1985, 82, 5131). Simultaneous use of two different supports with different densities (Tregear, Chemistry and Biology of Peptides, J. Meienhofer, ed., Ann Arbor Sci. Publ., Ann Arbor, 1972 pp. 175-178). Combination of reaction vessels through manifolds (Gorman, Anal. Biochem., 1984, 136, 397). Multicolumn solid-phase synthesis (eg, Krchnak, et al., Int. J. Peptide Protein Res., 1989, 33, 209), and Holm and Meldal. In "Proceedings of the 20th European Peptide Symposium ", G. Jung and E. Bayer, eds., Walter de Gruyter & Co., Berlin, 1989 pp. 208-210). Cellulose paper (Eichler, et al., Collect. Czech. Chem. Commun., 1989, 54, 1746). Support-mediated synthesis of peptides has also been reported (Synthetic Peptides: A User's Guide, Gregory A. Grant, Ed. Oxford University Press 1992; US-A-4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,132,418; 4,725,677 and Re-34,069).

Support-binding oligonucleotide synthesis relies on the sequential addition of nucleotides to one end of the growth chain. Typically, the first nucleoside (having a protecting group on any exocyclic amine functionality present) is attached to a suitable glass bead support and the activated phosphite compound (typically nucleotide phosphoramidite, also With appropriate protecting groups) are added in stages to stretch the growth oligonucleotides. Additional methods for solid-phase synthesis are described in Caruthers, US Pat. No. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; And 5,132,418; And US Pat. No. 4,725,677 to Koster and Re. 34,069.

Commercially available devices routinely used for the synthesis of oligomeric compounds and related compounds based on support media are sold by various vendors, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be used. Suitable solid phase techniques, including automated synthesis techniques, are described in [F. Eckstein (ed.), Oligonucleotides and Analogues, a Practical Approach, Oxford University Press, New York (1991).

In general, the phosphorus protecting group (pg) is O or S or an alkoxy or alkylthio group having a β-removable group of the formula —CH ₂ CH ₂ —G _W , wherein G _w is an electron withdrawing group. Suitable examples of pg applicable for use with the present invention include US Pat. No. 4,415,732 to Caruthers; 4,458,066; 4,500,707; 4,668,777; 4,973,679; And 5,132,418; And US Patent 4,725,677 to Koester and Re. Included are those described in 34,069. In general, alkyl or cyanoethyl attracting groups are preferred because methyl or cyanoethyl phosphorus protecting groups are generally incorporated into commercial phosphoramidites.

The method of removing pg depends on the particular pg to be removed. β-removable groups, such as those disclosed in the patent of Koster et al., are generally removed from weak base solutions, whereby acidic β-hydrogen is extracted by rearrangement and the -CH ₂ CH ₂ -G _w groups are removed, thereby Acrylo-compound CH ₂ = CH-G _w is formed. In contrast, alkyl groups are generally removed by nucleophilic attack on the α-carbons of alkyl groups. Such PGs are described in the patents of Carurthers et al. As cited herein.

Those skilled in the art will appreciate that oxidation of P (III) to P (V) can be carried out with a variety of reagents. Those skilled in the art will also recognize that P (V) species may be present as phosphate triesters, phosphorothioate diesters, or phosphorodithioate diesters. Each type of P (V) bond has the uses and advantages as described herein. Thus, the term “oxidizing agent” should be understood broadly as being any reagent capable of converting P (III) species (eg phosphite) to P (V). Thus the term "oxidant" includes "sulfurizing agent" which is also considered to have the same meaning as "thiation reagent". Unless otherwise modified, oxidation refers to the introduction of oxygen or sulfur with a concomitant increase in the P oxidation state from III to V. If it is important to indicate that the oxidant introduces oxygen into the P (III) species to become a P (V) species, then the oxidant will be referred to herein as an “oxygen-introducing oxidation reagent”.

Oxidation reagents (ie, oxygen-induced oxidation reagents) for making phosphate diester bonds under the phosphoramidite protocol are described, for example, in Carurthers et al. And Koester et al. It is. Examples of sulfiding reagents used to synthesize oligonucleotides containing phosphorothioate bonds include elemental sulfur, dibenzoyltetrasulfide, 3-H-1,2-benzidithiol-3-one 1,1-dioxide (Also known as bocaze reagents), tetraethylthiuram disulfide (TETD), and bis (O, O-diisopropoxy phosphinothioyl) disulfide (also known as Stec reagent) Included. Oxidation reagents for making phosphorothioate diester bonds are described by Cole et al. Phenylacetyldisulfide (PADS) as described in US Pat. No. 6,242,591. In some embodiments of the invention, phosphorothioate diesters and phosphate diester bonds may be alternated between sugar subunits. In another embodiment of the invention, phosphorothioate bonds may be used alone. In some embodiments, the thithiatization reagent may be dithiuram disulfide. See US Pat. No. 5,166,387 for the disclosure of some suitable dithiuram disulfides. It was unexpectedly found that one dithiuram disulfide can be used with standard capping reagents so that capping and oxidation can be performed in the same step. This is in contrast to standard oxidation reagents, such as bocage reagents, in which capping and oxidation have to occur in separate steps and generally involves column washing between the steps.

The 5′-protecting group bg or T ′ is orthogonal to the protecting group used to protect the nucleobase and, where appropriate, to the 2′-O-protecting group, as well as to the 3′-linker to the solid support medium. In some embodiments of the invention, the 5'-protecting group is acid degradable. In some embodiments according to the present invention, the 5′-protecting group is selected from optionally substituted trityl groups and optionally substituted pisyl groups. In some embodiments, the pixyl group is substituted with one or more substituents selected from alkyl, alkoxy, halo, alkenyl and alkynyl groups. In some embodiments, the trityl group is substituted with about 1 to about 3 alkoxy groups, especially about 1 to about 3 methoxy groups. In certain embodiments of the invention, the trityl group is substituted at the 4- and (if applicable) 4'-positions with one or two methoxy groups. Particularly acceptable trityl groups are 4,4'-dimethoxytrityl (DMT or DMTr).

In the context of the present invention, the term “reagent push” means the volume of solvent substantially free of any active compound (ie, reagents, activators, by-products, or other substances other than solvents), followed by reagents This volume of solvent is introduced into the column for the purpose of pushing the reagent solution onto or through the column prior to the solution and for the effect of pushing it. The reagent push need not be the entire column volume, but in some cases may comprise more than one times the column volume. In some embodiments, the reagent pushing at least removes the minimum volume necessary to substantially remove reagents, by-products, and / or activators from the cross section of the column immediately preceding the front formed by the reagent solution used for the immediate synthesis step. Include. If the concentration of the compound in the cross section of the column in which the subsequent reagent solution front is located is low enough to not substantially affect the activity of the subsequent reagent solution, the active compound is considered to have been substantially removed, regardless of whether it is a reagent, by-product or activator. . Those skilled in the art will appreciate that the volume of solvent required for "reagent push" will vary depending on the solvent, solubility in solvents such as reagents, activators, by-products, etc., on the column, amounts of reagents, activators, by-products, etc. to be removed from the column, and the like. Will recognize. Selecting a suitable volume for each reagent push is considered to be within the skill of one in the art, in particular with reference to the following examples.

As used herein, "column wash" has the same meaning as "reagent push" unless otherwise modified. In some embodiments of the present invention, column washing may mean that at least one time of the column volume is allowed to pass through the column before subsequent reagent solution is applied to the column. If the column volume (CV) of the column wash is specified, this indicates that a volume of solvent equivalent to the internal volume of the unfilled column is used for the column wash.

In the context of the present invention, the washing solvent is a solvent substantially free of the active compound applied to the column between the synthesis steps. A "washing step" is a step where a washing solvent is applied to a column. Both "push reagent" and "column cleaning" are included within this definition of "washing step".

The washing solvent can be a pure compound or a mixture of compounds, and the solvent can dissolve the active compound.

In some embodiments according to the present invention, the washing solvent used in one washing step may comprise some percentage of acetonitrile not exceeding 50% v / v.

If desired, the order of capping and oxidation steps can be reversed. That is, the capping may precede or follow the oxidation. In addition, with the selection of the appropriate thithiation reagent, the oxidation and capping steps can be combined in a single step. For example, it was unexpectedly found that capping with acetic anhydride can be performed in the presence of N, N'-dimethyldithiuram disulfide.

Various solvents can be used in the oxidation reaction. Suitable solvents are identified in the patents of Carurthers et al. And Koester et al. Cited herein. The patent in Cole et al. Describes acetonitrile as a solvent for phenylacetyldisulfide. Other suitable solvents include toluene, xanthene, dichloromethane and the like.

Reagents for cleaving oligonucleotides from supports are described, for example, in the patents of Carurthers et al. And Koester et al. As cited herein. When the phosphorus protecting group is 0-CH ₂ CH ₂ CN, cleavage of oligonucleotides containing thymidine (T) nucleotides in the presence of alkylated amines, such as triethylamine, is considered a good practice, whereby cyano-ethyl This is because the production of purified thymidine nucleotides (CNET) is currently known to be prevented. The prevention of CNET adducts is generally described in US Pat. No. 6,465,628, which is incorporated herein by reference, in particular in the examples of columns 20-30, which is specifically incorporated by reference.

Oligonucleotides can be worked up by standard procedures known in the art, such as size exclusion chromatography, high performance liquid chromatography (eg reverse phase HPLC), fractional precipitation, and the like. In some embodiments according to the invention, the 5′-OH protecting group is still present on the final nucleoside when the oligonucleotide is cleaved in the solid support medium. This so-called DMT-on (or trityl-on) oligonucleotide is then subjected to chromatography, the DMT group is removed by treatment with an organic acid, and then the oligonucleotide is desalted and further purified, resulting in final The product is formed.

The 5′-hydroxyl protecting group can be any group that is selectively removed under appropriate conditions. In particular, 4,4'-dimethoxytriphenylmethyl (DMT) groups are 5'- because they are readily cleaved under acidic conditions (for example in the presence of dichloroacetic acid (DCA), trichloroacetic acid (TCA), or acetic acid). It is a preferred group for protection in position. Removal of DMT from support-binding oligonucleotides is generally performed with DCA (eg, about 3 to about 10% DCA (v / v) in a suitable solvent). Removing oligonucleotides after cleavage from the support is generally performed with acetic acid.

As described herein, oligonucleotides can be prepared as chimeras with other oligomeric moieties. In the context of the present invention, the term "oligomeric compound" refers to a polymeric structure capable of hybridizing with a region of a nucleic acid molecule, and an "oligomeric moiety" refers to a portion of such oligomeric compound. Oligomeric compounds include oligonucleotides, oligonucleosides, oligonucleotide analogs, modified oligonucleotides and oligonucleotide mimetics. Oligomeric compounds may be linear or circular and may comprise branches. It may be single stranded or double stranded, and may comprise an overhang if double stranded. Generally, oligomeric compounds comprise a backbone to which monomeric subunits are linked, with each linked monomeric subunit attached directly or indirectly to a heterocyclic base moiety. The linkages, monomeric subunits and heterocyclic base moieties that link the monomeric subunits may differ from structure to structure, resulting in oligomeric, including hemimers, gapmers and chimeras. Allow multiple motifs to occur in the compound. As is known in the art, nucleosides are base-sugar combinations. The base portion of the nucleoside is generally a heterocyclic base moiety. The two most common classes of such heterocyclic bases are purines and pyrimidines. In the context of the present invention, the term “oligonucleoside” refers to nucleosides linked via internucleoside linkages that do not have a phosphorus atom. Internucleoside linkages of this type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more short chain heteroatoms and one or more short chain heterocyclic types. Such internucleoside linkages include siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkenyl, sulfamate; Methyleneimino, methylenehydrazino, sulfonates, sulfonamides, amides, and others having mixed N, O, S, and CH ₂ component moieties.

Syntheses for the synthesis of the replacement internucleoside linkages described above are described in US Pat. 5,034,506; 5,124,047; 5,278,302; 5,321,131; 5,519,126; 4,469,863; 5,455,233; 5,214,134; 5,470,967; 5,434,257. Further background information regarding internucleoside linkages is given in WO 91/08213; WO 90/15065; WO 91/15500; WO 92/20822; WO 92/20823; WO 91/15500; WO 89/12060; EP 216860; PCT / US 92/04294; PCT / US 90/03138; PCT / US 91/06855; PCT / US 92/03385; PCT / US 91/03680; US patent application 07 / 990,848; 07,892,902; 07 / 806,710; 07 / 763,130; 07 / 690,786; Stirchak, E. P., et al., Nucleic Acid Res., 1989, 17, 6129-6141; Hewitt, J.M., et al., 1992, 11, 1661-1666; Sod, A., et al., J. Am. Chem. Soc, 1990, 112, 9000-9001; Vasur, JJ. et al., J. Amer. Chem. Soc, 1992, 114, 4006-4007; Musichi, B., et al., J. Org. Chem., 1990, 55, 4231-4233; Reynolds, R. C., et al., J. Org. Chem., 1992, 57, 2983-2985; Mertes, M. P., et al., J. Med. Chem., 1969, 12, 154-157; Mungall, W. S., et al., J. Org. Chem., 1977, 42, 703-706; Stirchak, E. P., et al., J. Org. Chem., 1987, 52, 4202-4206; Coull, J. M., et al., Tet. Lett., 1987, 28, 745; And Wang, H., et al., Tet. Lett., 1991, 32, 7385-7388.

Phosphoramidites used for the synthesis of oligonucleotides are available from various commercial sources (Glen Research (Sterling, Virginia); Amersham Pharmacia Biotech Inc. (Piscataway, New Jersey); Cruachem Inc. (Aston, Pennsylvania); Chemgenes Corporation (Waltham, Massachusetts); Proligo LLC (Boulder, Colorado); PE Biosystems (Foster City, California); Beckman Coulter Inc. (Fullerton, California). These marketers generally sell high purity phosphoramidite with a purity of better than 98%. Those that do not provide batch purity for all amidites sold will include the assay in most cases, and each lot purchased provides at least the purity of the particular phosphoramid purchased. Commercially available phosphoramidites are mostly prepared for automated DNA synthesis, and therefore for immediate use to synthesize oligonucleotides of the desired sequence. Phosphoramidites are prepared, for example, by methods described in Carurthers et al. (US Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418) and Koester et al. (US Pat. No. 34,069). can do.

Double stranded oligonucleotides, such as double stranded RNA, can be constructed according to the methods of the invention, as described herein. In the case of RNA synthesis, it is necessary to protect the 2'-OH of the amidite reagent with an appropriate removable protecting group. Suitable protecting groups for 2'-OH are described in US Pat. Nos. 6,008,400, 6,111,086 and 5,889,136. Particularly suitable 2′-protecting groups for RNA synthesis are ACE protecting groups as described in US Pat. No. 6,111,086. In some embodiments, it is considered advantageous to use different 5′-protecting groups for the amidite used in RNA synthesis. Suitable 5'-protecting groups are described in US Pat. No. 6,008,400. Particularly suitable 5′-protecting groups are trimethylsilyloxy (TMSO) groups as taught in US Pat. No. 6,008,400. See, eg, Example 1, columns 10-13. Each strand of the double stranded RNA can be separately synthesized and then annealed to form double stranded (duplex) oligonucleotides.

Oligonucleotide Uses

Exemplary preferred antisense compounds include DNA or RNA sequences comprising eight or more consecutive nucleobases from the 5'-terminus of one of the exemplary preferred antisense compounds (the remaining nucleobases are the target nucleic acid. Is a continuous stretch of identical DNA or RNA starting immediately upstream of the 5'-end of an antisense compound that can hybridize specifically to and continuing until the DNA or RNA contains about 8 to about 80 nucleobases) . Similarly, preferred antisense compounds are represented by DNA or RNA sequences comprising 8 or more consecutive nucleobases from the 3'-terminus of one of the exemplary preferred antisense compounds (the remaining nucleobases are specific for the target nucleic acid). A continuous stretch of the same DNA or RNA starting immediately downstream of the 3'-end of the hybridizable antisense compound and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Once given the empirically derived preferred antisense compounds described herein, those skilled in the art will be able to identify additional preferred antisense compounds without undue experimentation.

Antisense and other compounds of the invention that hybridize to a target to inhibit expression of the target are identified through experiments, and representative sequences of these compounds are identified herein as preferred embodiments of the invention. While particular sequences of antisense compounds are described herein, those skilled in the art will recognize that they describe and describe particular embodiments within the spirit of the invention. Additional preferred antisense compounds can be identified by one skilled in the art.

Specific examples of preferred antisense compounds useful in the present invention include oligonucleotides containing modified backbone or non-natural nucleoside interbonds. As defined herein, oligonucleotides having modified backbones include those in which the phosphorus atoms are retained in the backbone and those in which the phosphorus atoms are not in the backbone. For the purposes of this specification and as often referenced in the art, modified oligonucleotides that do not have a phosphorus atom in the nucleoside liver backbone are also considered to be oligonucleosides.

RNA Degrading enzyme H-dependent antisense

One method of inhibiting expression of certain genes involves using oligonucleotides or oligonucleotide analogs as "antisense" agents. Antisense techniques involve directing an oligonucleotide, or analog thereof, to a specific target messenger RNA (mRNA) sequence. The interaction of exogenous "antisense" molecules with endogenous mRNA modulates transcription by a variety of pathways. Such pathways include transcription retardation, RNAase H recruitment, and RNAi (eg siRNA). Antisense technology allows specific protein activity to be modulated in a relatively predictable manner.

The present invention will be further understood by reference to the following non-limiting and illustrative examples, which can generally be carried out by the methods described above.

Example  One

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethyl styrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 7 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  2

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethylstyrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 15 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  3

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethylstyrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 20 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  4

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethylstyrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. The reactor was then heated to 80 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  5

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethylstyrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. The reactor was then heated to 85 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  6

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (250 g), 55% -divinylbenzene (45% -ethylstyrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexanol An organic solution consisting of (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  7

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (110 g), 55% -divinylbenzene (45% -ethylstyrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. The reactor was then heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  8

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethylstyrene) (15 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  9

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethylstyrene) (30 g), acetoxystyrene (20 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads are dispersed in acetone, then sieved and filtered under vacuum.

Example  10

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethylstyrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (40 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at fixed speed (250 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  11

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethyl styrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at a fixed speed (350 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  12

Deionized water (1500 mL) and polyvinyl alcohol (40 g) were placed in a reaction vessel (2 L) equipped with a mechanical stirrer, a condenser and a nitrogen inlet. The reaction was maintained under a nitrogen atmosphere throughout the entire polymerization process. Styrene (190 g), 55% -divinylbenzene (45% -ethylstyrene) (30 g), acetoxystyrene (10 g), benzoyl peroxide (4 g), isooctane (90 g), 2-ethylhexa An organic solution consisting of knol (200 g) was added to the reaction vessel. The mixture was stirred at a fixed speed (450 rpm) to produce the desired bead size distribution. Thereafter, the reactor was heated to 75 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and dried under vacuum. Deionized water (300 mL), ethanol (1000 mL) and dried beads were placed in a reaction vessel (2 L) equipped with a mechanical stirrer (200 rpm) and a condenser. Thereafter, the reactor was heated to 70 ° C. After 12 hours, the motor was stopped and the beads formed were filtered and washed with deionized water and acetone. The beads were dispersed in acetone, then sieved and filtered under vacuum.

Example  13: 5'-O- DMT Thymidine Loading of -3'-O-succinate to a solid support

Loading of the protected nucleosides was carried out using the solid support obtained from Experiment 1 under standard conditions. Solid support, Hunig base (12 equiv), HBTU activator (4 equiv), DMT T nucleoside succinate as triethyl ammonium salt (2.0 equiv) were placed in a round bottom flask and sealed at room temperature It was shaken mechanically for 10 hours. The support was then washed with acetonitrile (100 mL) and dried. Thereafter, a mixture of Cap A and Cap B solutions (20 mL each) used for oligomerization was added to the solid support, followed by the addition of a catalytic amount of 4-dimethylaminopyridine and mechanically shaken overnight. The support was washed with acetonitrile (200 mL), methanol (100 mL) and finally anhydrous ether (200 mL). Finally the support was completely dried and stored.

Example  14: 5'-O- DMT - N4 - Benzoyl -2'- Deoxycytidine Loading of -3'-O-succinate to a solid support

Loading of the protected nucleosides was carried out using the solid support obtained from Experiment 1 under standard conditions. Solid support, Hunig base (12 equiv), HBTU activator (4 equiv), 5'-0-DMT-N4-benzoyl-2'-deoxycytidine-3'-0-succinate as triethyl ammonium salt (2.0 equiv) was placed in a round bottom flask, which was sealed and mechanically shaken for 10 hours at room temperature. The support was then washed with acetonitrile (100 mL) and dried. Thereafter, a mixture of Cap A and Cap B solutions (20 mL each) used for oligomerization was added to the solid support, followed by the addition of a catalytic amount of 4-dimethylaminopyridine and mechanically shaken overnight. The support was washed with acetonitrile (200 mL), methanol (100 mL) and finally anhydrous ether (200 mL). Finally the support was completely dried and stored.

Example  15: 5'-O- DMT - N6 - Benzoyl -2'- Deoxyadenosine Loading of -3'-O-succinate to a solid support

Loading of the protected nucleosides was carried out using the solid support obtained from Experiment 1 under standard conditions. Solid support, Hunig base (12 equiv), HBTU activator (4 equiv), 5'-O-DMT-N6-benzoyl-2'-deoxyadenosine-3'-O-succinate as triethyl ammonium salt ( 2.0 equivalents) was placed in a round bottom flask, which was sealed and mechanically shaken for 10 hours at room temperature. The support was then washed with acetonitrile (100 mL) and dried. Thereafter, a mixture of Cap A and Cap B solutions (20 mL each) used for oligomerization was added to the solid support, followed by the addition of a catalytic amount of 4-dimethylaminopyridine and mechanically shaken overnight. The support was washed with acetonitrile (200 mL), methanol (100 mL) and finally anhydrous ether (200 mL). Finally the support was completely dried and stored.

Example  16: 5'-O- DMT - N2 - Isobutyryl -2'- Deoxyguanosine Loading of -3'-O-succinate to solid retardation

Loading of the protected nucleosides was carried out using the solid support obtained from Experiment 1 under standard conditions. 5'-O-DMT-N2-isobutyryl-2'-deoxyguanosine-3'-O- as a solid support, Hunig base (12 equiv), HBTU activator (4 equiv), triethyl ammonium salt Succinate (2.0 equiv) was placed in a round bottom flask, sealed and mechanically shaken for 10 hours at room temperature. The support was then washed with acetonitrile (100 mL) and dried. Thereafter, a mixture of Cap A and Cap B solutions (20 mL each) used for oligomerization was added to the solid support, followed by the addition of a catalytic amount of 4-dimethylaminopyridine and mechanically shaken overnight. The support was washed with acetonitrile (200 mL), methanol (100 mL) and finally anhydrous ether (200 mL). Finally the support was completely dried and stored.

Example  17: 5'-O- DMT -2'-O- Methoxyethyl -5- Methyluridine Loading of -3'-O-succinate to a solid support

Loading of the protected nucleosides was carried out using the solid support obtained from Experiment 1 under standard conditions. Solid support, Hunig base (12 equiv), HBTU activator (4 equiv), 5'-0-DMT-2'-0-methoxyethyl-5-methyluridine-3'-0 as triethyl ammonium salt -Succinate (2.0 equiv) was placed in a round bottom flask, which was sealed and mechanically shaken for 10 hours at room temperature. The support was then washed with acetonitrile (100 mL) and dried. Thereafter, a mixture of Cap A and Cap B solutions (20 mL each) used for oligomerization was added to the solid support, followed by the addition of a catalytic amount of 4-dimethylaminopyridine and mechanically shaken overnight. The support was washed with acetonitrile (200 mL), methanol (100 mL) and finally anhydrous ether (200 mL). Finally the support was completely dried and stored.

Example  18: 5'-O- DMT - N4 - Benzoyl -2'-O- Methoxyethylcytidine Loading of -3'-O-succinate to a solid support

Loading of the protected nucleosides was carried out using the solid support obtained from Experiment 1 under standard conditions. Solid support, Hunig base (12 equiv), HBTU activator (4 equiv), 5'-0-DMT-N4-benzoyl-2'-0-methoxyethylcytidine-3'-O as triethyl ammonium salt -Succinate (2.0 equiv) was placed in a round bottom flask, which was sealed and mechanically shaken for 10 hours at room temperature. The support was then washed with acetonitrile (100 mL) and dried. Thereafter, a mixture of Cap A and Cap B solutions (20 mL each) used for oligomerization was added to the solid support, followed by the addition of a catalytic amount of 4-dimethylaminopyridine and mechanically shaken overnight. The support was washed with acetonitrile (200 mL), methanol (100 mL) and finally anhydrous ether (200 mL). Finally the support was completely dried and stored.

Example  19: 5'-O- DMT - N6 - Benzoyl -2'-O- Methoxyethyladenosine Loading of -3'-O-succinate to a solid support

Loading of the protected nucleosides was carried out using the solid support obtained from Experiment 1 under standard conditions. Solid support, Hunig base (12 equiv), HBTU activator (4 equiv), 5'-0-DMT-N6-benzoyl-2'-0-methoxyethyladenosine-3'- as triethyl ammonium salt O-succinate (2.0 equiv) was placed in a round bottom flask and sealed and mechanically shaken for 10 hours at room temperature. The support was then washed with acetonitrile (100 mL) and dried. Thereafter, a mixture of Cap A and Cap B solutions (20 mL each) used for oligomerization was added to the solid support, followed by the addition of a catalytic amount of 4-dimethylaminopyridine and mechanically shaken overnight. The support was washed with acetonitrile (200 mL), methanol (100 mL) and finally anhydrous ether (200 mL). Finally the support was completely dried and stored.

Example  20: 5'-O- DMT - N2 - Isobutyryl -2'-O- Methoxyethylguanosine Loading of -3'-O-succinate to a solid support

Loading of the protected nucleosides was carried out using the solid support obtained from Experiment 1 under standard conditions. Solid support, Hunig base (12 equiv), HBTU activator (4 equiv), 5'-0-DMT-N2-isobutyryl-2'-0-methoxyethylguanosine-3 'as triethyl ammonium salt -O-succinate (2.0 equiv) was placed in a round bottom flask and sealed and mechanically shaken for 10 hours at room temperature. The support was then washed with acetonitrile (100 mL) and dried. Thereafter, a mixture of Cap A and Cap B solutions (20 mL each) used for oligomerization was added to the solid support, followed by the addition of a catalytic amount of 4-dimethylaminopyridine and mechanically shaken overnight. The support was washed with acetonitrile (200 mL), methanol (100 mL) and finally anhydrous ether (200 mL). Finally the support was completely dried and stored.

Example  21: 5'-d (fully deformed) TCC - CGC - CTG - TGA - CAT - GCA - TT ) -3 ' Phosphorothio  The 20-mer  synthesis

Synthesis of the sequence was carried out on Amersham Biosciences' Akta 100 DNA / RNA synthesizer using cyanoethyl phosphoramidite and DMT thymidine-derivatized solid support prepared as above on a scale of about 420 micromolar. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  22: 5'-d (fully transformed) GTT - CTC - GCT - GGT - GAG - TTT - CA ) -3 ' Phosphoro Thioate 20-mer  synthesis

Using cyanoethyl phosphoramidite and DMT N6-benzoyl-2'-deoxyadenosine-derivatized solid support prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of the sequence was performed. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  23: 5'-d (completely modified) GCC - CAA - GCT - GGC - ATC - CGT - CA ) -3 ' Phosphoro Thioate 20-mer  synthesis

Using cyanoethyl phosphoramidite and DMT N6-benzoyl-2'-deoxyadenosine-derivatized solid support prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of the sequence was performed. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  24: 5'-d (completely modified) TCC - GTC - ATC - GCT - CCT - CAG - GG ) -3 ' Phosphorothioate 20-mer  synthesis

Cyanoethyl phosphoramidite and DMT N2-isobutyryl-2'-deoxyguanosine-derivatized solid support prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer at a scale of about 420 micromolar The synthesis of these sequences was performed. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  25: 5'-d (completely modified) GTT - CTC - GCT - GGT - GAG - TTT - CA ) -3 ' Phosphorothioate 20-mer  synthesis

Using cyanoethyl phosphoramidite and DMT N6-benzoyl-2'-deoxyadenosine-derivatized solid support prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of the sequence was performed. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  26: 5 '-[2'-O- completely modified Methoxyethyl -( TGTG ] -d ( CTA - TTC - TGT -G-)-[2'-O-Me Oxyet Teal- ( AATT ] -3 ' Phosphorothioate 18-mer  synthesis

Cyanoethyl phosphoramidite and 5'-O-DMT-2'-O-methoxyethyl-5-methyluri prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of this sequence was performed using a Dean-derivatized solid support. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  27: fully modified 5 '-[2'-O- Methoxyethyl -( CAGC ] -d ( AGC - AGA - GTC - TTC -A-)-[2'-O-Me Talk Cyethyl- ( TCAT ] -3 ' Phosphorothioate 21-mer  synthesis

Cyanoethyl phosphoramidite and 5'-O-DMT-2'-O-methoxyethyl-5-methyluri prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of this sequence was performed using a Dean-derivatized solid support. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  28: 5 '-[2'-O- completely modified Methoxyethyl -( GCTCC ] -d ( TTC - CAC - TGA -T)-[2'-O-Me Oxyet Teal- ( CCTGC ] -3 ' Phosphorothioate 20-mer  synthesis

Cyanoethyl phosphoramidite and 5'-O-DMT-2'-O-methoxyethyl-5-methyl prepared as described above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of this sequence was performed using a Tidine-derivatized solid support. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  29: 5 '-[2'-O- completely modified Methoxyethyl -( GCTCC ] -d ( TTC - CAC - TGA -T)-[2'-O-Me Oxyet Teal- ( CCTGC ] -3 ' Phosphorothioate 20-mer  synthesis

Cyanoethyl phosphoramidite and 5'-O-DMT-2'-O-methoxyethyl-5-methyl prepared as described above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of this sequence was performed using a Tidine-derivatized solid support. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  30: 5 '-[2'-O- completely deformed Methoxyethyl -( GCCTC ] -d ( AGT - CTG - CTT -C)-[2'-O-meth Oxyet Teal- ( GCACC ] -3 ' Phosphorothioate 20-mer  synthesis

Cyanoethyl phosphoramidite and 5'-O-DMT-2'-O-methoxyethyl-5-methyl prepared as described above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of this sequence was performed using a Tidine-derivatized solid support. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Sulfation was carried out for 2 minutes using a 0.2 M solution of phenylacetyl disulfide in acetonitrile: 3-picolin (1: 1 v / v). At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphorothioate oligonucleotides.

Example  31: 5'-d ( TCC - CGC - CTG - TGA - CAT - GCA - TT ) -3 ' Phosphate Diester 20-mer  synthesis

Synthesis of the sequence was carried out on Amersham Biosciences' Akta 100 DNA / RNA synthesizer using cyanoethyl phosphoramidite and DMT thymidine-derivatized solid support prepared as above on a scale of about 420 micromolar. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Oxidation was performed for 2 minutes using standard iodine solution. At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphate oligonucleotide.

Example  32: 5'-d ( GTT - CTC - GCT - GGT - GAG - TTT - CA ) -3 ' Phosphate Diester 20-mer  synthesis

Using cyanoethyl phosphoramidite and DMT N6-benzoyl-2'-deoxyadenosine-derivatized solid support prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of the sequence was performed. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Oxidation was performed for 2 minutes using standard iodine solution. At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphate oligonucleotide.

Example  33: 5'-d ( GCC - CAA - GCT - GGC - ATC - CGT - CA ) -3 ' Phosphate Diester 20-mer  synthesis

Using cyanoethyl phosphoramidite and DMT N6-benzoyl-2'-deoxyadenosine-derivatized solid support prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of the sequence was performed. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Oxidation was performed for 2 minutes using standard iodine solution. At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphate oligonucleotide.

Example  34: 5'-d ( TCC - GTC - ATC - GCT - CCT - CAG - GG ) -3 ' Phosphate Diester 20-mer  synthesis

Cyanoethyl phosphoramidite and DMT N2-isobutyryl-2'-deoxyguanosine-derivatized solid support prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer at a scale of about 420 micromolar The synthesis of these sequences was performed. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Oxidation was performed for 2 minutes using standard iodine solution. At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphate oligonucleotide.

Example  35: 5'-d ( GTT - CTC - GCT - GGT - GAG - TTT - CA ) -3 ' Phosphate 20-mer  synthesis

Using cyanoethyl phosphoramidite and DMT N6-benzoyl-2'-deoxyadenosine-derivatized solid support prepared as above on an Amersham Biosciences Akta 100 DNA / RNA synthesizer on a scale of about 420 micromolar Synthesis of the sequence was performed. Trityl removal was performed using 10% dichloroacetic acid (volume / volume) in toluene. Oxidation was performed for 2 minutes using standard iodine solution. At the end of the synthesis, the support was washed with acetonitrile, cleaved and deprotected at 55 ° C. for 12 hours with ammonium hydroxide. The crude material was purified in a conventional manner to yield the desired phosphate oligonucleotide.

Example  36: general procedure for the synthesis of oligonucleotides

2.2mM synthesis was performed according to the following procedure:

1. Drain 11.0 g of 200 μm loading support into a 35 mm flow-through column;

2. Add about 150 mL of toluene to the column and allow the support to swell for several minutes. The swollen support should be about 5.2 cm from the bottom plate of the column to the top of the support bed.

3. Lower the column's top net adapter to about 7.2 cm, creating a 2 cm gap between the support bed and the column top plate.

4. Secure the column locking mechanism.

5. Solid phase oligonucleotide synthesis is carried out using nucleoside phosphoramidites to produce oligonucleotides having phosphodiester, phosphorothioate, or phosphorodithioate internucleoside linkages.

The synthesis of step 5 involves attaching the first nucleoside to the support via a linker (e.g. a unilinker) using standard reagents, provided that the support is not derivatized to contain such a first synthetic unit, It may include. Alternatively, if the support has been derivatized beforehand, the derivatized support is loaded onto the column as described above and the synthesis proceeds with the addition of phosphoramidite synthesizing units. In addition, if the support (not derivatized or derivatized) has been swollen before loading onto the column, the swollen support is loaded to allow for the above-mentioned 2 cm spacing.

Example  37: Polymer Bead  synthesis

To determine the effect of various experimental parameters on the properties of the beads, several beads were produced under different manufacturing conditions and their physical properties were tested. The results are summarized in the table below.

Those skilled in the art will understand that numerous changes and modifications can be made to preferred embodiments of the invention, and such changes and modifications can be made without departing from the spirit of the invention. Accordingly, it is intended that the appended claims cover all such variants as fall within the true spirit and scope of this invention.

Published publications that include each patent, patent application, and book mentioned in this patent document are intended to be incorporated herein in their entirety by reference.

This application claims priority of US patent provisional application 60 / 606,873, filed September 2, 2004, the entire disclosure of which is incorporated herein by reference.

Claims (32)

(a) providing an organic phase; And (b) contacting the organic phase with an aqueous phase at a temperature of 25 ° C. to 95 ° C. In the method for producing a polymeric bead comprising: The organic phase comprises an olefin monomer, a crosslinking monomer, a functionalized monomer, an initiator, an organic solvent; The olefin monomer comprises at least one aryl-vinyl compound; The crosslinking monomer is an olefinic crosslinking monomer having two non-conjugated vinyl groups attached to an aromatic moiety; The functionalized monomer is an olefinic monomer having a protected hydroxyl group, The organic solvent comprises at least one liquid hydrocarbon, an alcohol having 5 to 12 carbon atoms, or both; The aqueous phase comprises water and a dispersing reagent; The weight percentage of the olefin monomer initially present in the monomer is from 60% to 96%; The weight percentage of crosslinking monomer initially present in the monomer is 3% to 9.9%; The weight percentage of the functionalized monomer initially present in the monomer is 1% to 20%; The weight percentage of monomers initially present in the organic phase is 33% to 67%; The weight percentage of organic solvent initially present in the organic phase is 33% to 67%; The weight percentage of the liquid hydrocarbon present in the organic solvent is 0% to 80%; The weight percentage of the alcohol having 5 to 12 carbon atoms initially present in the organic solvent is 20% to 100%; Wherein the weight percentage of the dispersing reagent initially present in the aqueous phase is between 0.01% and 20%. The method of claim 1, The weight percentage of the olefin monomer initially present in the monomer is from 75% to 94%; The weight percentage of crosslinking monomer initially present in the monomer is 4% to 9.9%; The weight percentage of the functionalized monomer initially present in the monomer is 2% to 10%; The weight percentage of monomers initially present in the organic phase is between 35% and 60%; The weight percentage of organic solvent initially present in the organic phase is between 40% and 65%; The weight percentage of hydrocarbons present in the organic solvent is 5% to 70%; The weight percentage of alcohol having 5 to 12 carbon atoms initially present in the organic solvent is 30% to 95%. The method of claim 1, The weight percentage of the olefin monomer initially present in the monomer is 82% to 91.5%; The weight percentage of crosslinking monomer initially present in the monomer is from 5.5% to 9.9%; The weight percentage of the functionalized monomer initially present in the monomer is 3% to 8%; The weight percentage of monomers initially present in the organic phase is between 40% and 50%; The weight percentage of organic solvent initially present in the organic phase is between 50% and 60%; The weight percentage of the liquid hydrocarbon present in the organic solvent is 10% to 60%; The weight percentage of the alcohol having 5 to 12 carbon atoms initially present in the organic solvent is 40% to 90%. delete The method of claim 1, wherein the olefin monomer comprises styrene, ethyl styrene, or both. delete The method of claim 1 wherein the aromatic moiety of the olefinic crosslinking monomer is a five or six membered aromatic ring. The method of claim 1 wherein the crosslinking monomer is divinylbenzene. delete delete The method of claim 1 wherein the functionalized monomer is acetoxystyrene. The method of claim 1 wherein the initiator is a stabilized peroxide or azo compound. 13. The method of claim 12 wherein the stabilized peroxide comprises benzoylperoxide. The method of claim 1, wherein the organic solvent comprises one or more liquid alkanes, benzene, toluene, xylene, alcohols having 5 to 12 carbon atoms, or a combination thereof. The method of claim 1, wherein the organic solvent comprises at least one octane, alcohol having 5 to 12 carbon atoms, or both. The method of claim 1, wherein the organic solvent comprises isooctane, 2-ethylhexanol, or both. The method of claim 1 wherein the dispersing reagent comprises polyalcohol. The method of claim 1 wherein the dispersing reagent comprises polyvinyl alcohol. delete The method of claim 1 wherein the organic phase and the aqueous phase are heated to a temperature of 70 ° C. to 85 ° C. 7. The method of claim 1 wherein the organic phase and the aqueous phase are heated to a temperature of 75 ° C. to 80 ° C. 7. Polymeric beads formed by the method of claim 1. The polymeric bead according to claim 22, wherein the loading capacity of the beads is 100 μmol per gram of beads to 350 μmol per gram of beads. The polymeric bead of claim 22 wherein said beads have an average particle size of 10-300 μm. The polymeric bead of claim 22, wherein the average pore size of the bead is 10-100 nm. The polymeric bead according to claim 22, wherein the specific surface area of the bead is 10-100 m 2 / g. A compound of formula (I) (I)

[Meal: G ₃ is O, S, CH ₂ , or NH; G ₅ is O, S, CH ₂ , CFH, CF ₂ , or -CH = CH-; G ₆ is O, S, CH ₂ , or NH; Each R ₂ ′ is H, OH, O-rg (where rg is a removable protecting group), 2′-substituent, or bridges with R ₄ ′; Each R ₃ ′ is H, a substituent, or forms a bridge with R ₄ ′; Each R ₄ ′ is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′; q is 0 or 1; R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′; Bx is a nucleobase; T 'is H or a removable protecting group; LL is a linking moiety; SS is the bead of claim 22]. A compound of formula II: ≪ RTI ID = 0.0 &

[Meal: m is an integer from 0 to 100; Each G ₁ is O or S; Each G ₂ is OH or SH; Each G ₃ is O, S, CH ₂ , or NH; Each G ₅ is O, S, CH ₂ , CFH, CF ₂ , or —CH═CH—; Each R ₂ ′ is H, OH, O-rg (where rg is a removable protecting group), 2′-substituent, or bridges together with R ₄ ′; Each R ₃ ′ is H, a substituent, or forms a bridge with R ₄ ′; Each R ₄ ′ is H, a substituent, or bridges with R ₂ ′, bridges with R ₃ ′, or bridges with R ₅ ′; Each q is 0 or 1; Each R ₅ ′ is H, a substituent, or forms a bridge with R ₄ ′; Each G ₆ is independently O, S, CH ₂ or NH; Each Bx is a nucleobase; pg is a removable phosphorus protecting group; T 'is a removable protecting group; LL is a linking moiety; SS is the bead of claim 22]. delete delete delete delete